Stochastic confinement to detect, manipulate, and utilize molecules and organisms

ABSTRACT

Methods of detecting organisms e.g. bacteria using stochastic confinement effects with microfluidic technologies involving plugs are provided. Signal amplification methods for the detection of molecules are also disclosed.

RELATED APPLICATIONS

The present patent document claims the benefit of the filing date under35 U.S.C. §119(e) of Provisional U.S. Patent Application Ser. Nos.60/962,426, filed Jul. 26, 2007, and 61/052,490 filed May 12, 2008,which are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by Grant No. 0526693 awarded by theNational Science Foundation (NSF) CRC and under grant numbers EB01903,GM075827, and GM074961 awarded by the National Institutes of Health(NIH). The government has certain rights in the invention.

BACKGROUND

Bacterial infections are a major health problem, leading to more than130,000 deaths from sepsis annually in the United States alone. (G. S.Martin, D. M. Mannino, S. Eaton and M. Moss, N. Engl. J. Med., 2003,348, 1546-1554) These deaths are often the result of nosocomial, orhospital acquired, infections and frequently involve drug resistantstrains of bacteria. (B. M. Farr, Curr. Opin. Infect. Dis., 2004, 17,317-322; G. J. Moran, A. Krishnadasan, R. J. Gorwitz, G. E. Fosheim, L.K. McDougal, R. B. Carey and D. A. Talan, N. Engl. J. Med., 2006, 355,666-674) In addition, bacteremia, the presence of bacteria in the blood,is one of the major causes of sepsis and generally requires a minimum ofa day or more to diagnose, increasing the chances of patient mortality.(S. D. Carrigan, G. Scott and M. Tabrizian, Clin. Chem., 2004, 50,1301-1314) Patient mortality rates further increase when inappropriateantimicrobial treatment is administered, which is estimated to occur in23-30% of cases. (S. D. Carrigan, G. Scott and M. Tabrizian, Clin.Chem., 2004, 50, 1301-1314)

Shortening the time necessary to detect and identify an effectiveantibiotic regimen to treat bacterial infections could significantlydecrease the mortality rate and reduce the cost of treating patientswith sepsis and other aggressive bacterial infections. (H. B. Nguyen, E.P. Rivers, F. M. Abrahamian, G. J. Moran, E. Abraham, S. Trzeciak, D. T.Huang, T. Osborn, D. Stevens and D. A. Talan, Ann. Emerg. Med., 2006,48, 28-54) However, attempts to reduce the assay time of traditionaldiagnosis and characterization techniques are impeded by the necessityto incubate bacterial specimens for hours to days to increase the celldensity of the sample to detectible levels. To overcome this challenge,new PCR-based detection methods enable diagnosis in the one to four hourtime frame. (S. Poppert, A. Essig, B. Stoehr, A. Steingruber, B. Wirths,S. Juretschko, U. Reischl and N. Wellinghausen, J. Clin. Microbiol.,2005, 43, 3390-3397; K. P. Hunfeld, Int. J. Med. Microbiol., 2007, 297,32-32.) However, these methods only provide a genetic profile of theinfecting bacterial species and lack the ability to directly test thebacteria's function, such as susceptibility to particular antibiotics.Although some types of antibiotic resistance have genetic markers, suchas the mecA gene for instance, (K. Murakami, W. Minamide, K. Wada, E.Nakamura, H. Teraoka and S. Watanabe, J. Clin. Microbiol., 1991, 29,2240-2244) genetic markers have not been identified for all antibioticresistant strains of bacteria. Therefore, antibiotic susceptibility ismore accurately determined by a functional assay, especially forbacterial strains with unknown resistance mechanisms.

There are many people (10⁵-10⁶) affected every year, and there is nomethod of detection that works. Early diagnosis of the presence and typeof bacteria in a patient's blood stream would help prevent the death ofmillions of people dying from sepsis. Currently, blood is drawn from apatient and cultures are done to grow the bacteria. However it usuallytakes days to weeks to grow enough bacteria to detect them, and by thetime they grow in the culture, they also grow in the patient, and thepatient becomes very sick.

The broth in the blood culture bottle is the first step in creating anenvironment in which bacteria will grow. It contains all the nutrientsthat bacteria need to grow. If the physician expects anaerobic bacteriato grow, oxygen will be kept out of the blood culture bottle; if aerobesare expected, oxygen will be allowed in the bottle.

The bottles are placed in an incubator and kept at body temperature.They are watched daily for signs of growth, including cloudiness or acolor change in the broth, gas bubbles, or clumps of bacteria. Whenthere is evidence of growth, the laboratory does a gram stain and asubculture. To do the gram stain, a drop of blood is removed from thebottle and placed on a microscope slide. The blood is allowed to dry andthen is stained with purple and red stains and examined under themicroscope. If bacteria are seen, the color of stain they picked up(purple or red), their shape (such as round or rectangular), and theirsize provide valuable clues as to what type of microorganism they areand what antibiotics might work best. To do the subculture, a drop ofblood is placed on a culture plate, spread over the surface, and placedin an incubator.

If there is no immediate visible evidence of growth in the bottles, thelaboratory looks for bacteria by doing gram stains and subcultures.These steps are repeated daily for the first several days andperiodically after that.

When bacteria grow, the laboratory identifies it using biochemical testsand the Gram stain. Sensitivity testing, also called antibioticsusceptibility testing is performed as well. The bacteria are testedagainst many different antibiotics to see which antibiotics caneffectively kill it.

All information is passed on to the physician as soon as it is known. Anearly report, known as a preliminary report, is usually available afterone day. This report will tell if any bacteria have been found yet, andif so, the results of the gram stain. The next preliminary report mayinclude a description of the bacteria growing on the subculture. Thelaboratory notifies the physician immediately when an organism is foundand as soon as sensitivity tests are complete. Sensitivity tests may becomplete before the bacteria are completely identified. The final reportmay not be available for five to seven days. If bacteria are found, thereport will include its complete identification and a list of theantibiotics to which the bacteria is sensitive.

What is needed is a faster and better method of detecting organisms,including improving the accuracy and decreasing the man-hours associatedwith standard blood culturing, and shortening the time necessary todetect and identify an effective antibiotic regimen to treat bacterialinfections.

BRIEF SUMMARY

In one embodiment, a method of detecting an organism is provided. Themethod comprises flowing at least two plugs in a carrier fluid through amicrochannel; introducing a sample optionally comprising the organisminto the first and second plugs; and analyzing the at least two plugsfor the detectable signal. Each plug comprises a plug fluid that issubstantially immiscible with the carrier fluid and the organismproduces a detectable signal.

In a second embodiment, a method of detecting bacteria in a patient isprovided. The method comprises flowing at least two plugs in a carrierfluid through a microchannel; introducing a patient sample optionallycomprising bacteria into the at least two plugs; and analyzing the atleast two plugs for the detectable signal. Each plug comprises a plugfluid that is substantially immiscible with the carrier fluid and thebacteria produce a detectable signal.

In a third embodiment, a method of detecting bacteria is provided. Themethod comprises flowing at least two plugs in a carrier fluid through amicrochannel; introducing a sample optionally comprising bacteria intothe first and second plugs, wherein the bacteria produce a detectablesignal; and analyzing the plugs for the detectable signal. Each plugcomprises a plug fluid substantially immiscible with the carrier fluid.The first plug comprises a means for detecting a first species ofbacteria and the second plug comprises a means for detecting a secondspecies of bacteria different from the first species of bacteria.

In a fourth embodiment, a method of detecting bacteria is providedcomprising flowing at least two plugs in a carrier fluid through amicrochannel; introducing a sample optionally comprising bacteria intothe first and second plugs; and detecting the presence of bacteria boundbeads. Each plug comprises a plug fluid substantially immiscible withthe carrier fluid. The first plug comprises a first antibody bound bead,the first antibody bound bead comprising a first bead and a firstantibody that binds to a first bacteria. The second plug comprises asecond antibody bound bead, the second antibody bound bead comprising asecond bead and a second antibody that binds to a second bacteriadifferent than the first bacteria. The bacteria bind to the antibodybound beads to form bacteria bound beads.

In a fifth embodiment, a method of screening for antibiotic activity isprovided. The method comprises flowing at least two plugs in a carrierfluid through a microchannel. Each plug comprises a plug fluidsubstantially immiscible with the carrier fluid and an antibody boundbead, the antibody bound bead comprising a bead and an antibody thatbinds bacteria; introducing a sample which comprises bacteria into thefirst and second plugs; and detecting the presence of either antibodybound beads or bacteria bound beads. The first plug comprises a firstantibiotic candidate and the second plug comprises a second antibioticcandidate. The bacteria bind to the antibody bound beads to formbacteria bound beads.

In a sixth embodiment, a method of screening for antibiotic activity isprovided comprising flowing at least two plugs in a carrier fluidthrough a microchannel; introducing a sample which comprises bacteriainto the first and second plugs; detecting the presence of eitherantibody bound beads or bacteria bound beads. Each plug comprises a plugfluid substantially immiscible with the carrier fluid and an antibodybound bead, the antibody bound bead comprising a bead and an antibodythat binds bacteria. The first plug comprises a first antibioticcandidate and the second plug comprises a second antibiotic candidate.The bacteria bind to the antibody bound beads to form bacteria boundbeads.

In a seventh embodiment, a method of screening for antibiotic activityis provided comprising flowing at least two plugs in a carrier fluidthrough a microchannel; introducing a sample which comprises bacteriainto the first and second plugs; and detecting the presence of bacterialgrowth. Each plug comprises a plug fluid immiscible with the carrierfluid and media capable of supporting bacterial growth. The first plugcomprises a first antibiotic candidate and the second plug comprises asecond antibiotic candidate.

In an eighth embodiment, a method of detecting molecules is providedcomprising providing a first set of molecules that constitute anautocatalytic loop, capable of amplification of one of the components;providing a second set of molecules that modulate the autocatalytic loopwhen the autocatalytic loop reacts with the target molecules; andanalyzing for the presence of the target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of detecting bacteria using stochasticconfinement of bacteria into plugs reduces detection time. (a) Aschematic drawing illustrating the increase in cell density resultingfrom the stochastic confinement of an individual bacterium in ananoliter-sized plug. (b) A schematic drawing illustrates theexperimental procedure to compare the detection of bacteria incubated innanoliter-sized plugs and bacteria incubated in a milliliter-scaleculture. (c) An illustrative graph of decreased detection time vs. thelog of the plug volume. (d) An illustrative graph of detection times vs.cell density for bacteria incubated in plugs (circles) and bacteriaincubated in 96 well plates (crosses) with similar initial celldensities.

FIG. 2. illustrates a method for screening many antibiotics against abacterial sample using a combination stochastic confinement withmicrofluidic hybrid and/or cartridge methods. (a) A schematic drawingillustrating the formation of plugs of bacteria, viability indicator,and antibiotic from a preformed array of plugs of different antibiotics.(b) An illustrative graph of fluorescence intensity of the control plugswith no antibiotic (+, blank1, positive control) and vancomycin (A, VCM,negative control). (c) An illustrative bar graph shows the results ofthe antibiotic screen against the Methicillin Resistant S. aureus(MRSA), indicating that this strain of MRSA was resistant to fourantibiotics, but sensitive to two. (d) An illustrative chart shows theagreement between the susceptibility profiles (S, sensitive and R,resistant) of MRSA determined by the plug-based microfluidic screen andthe control susceptibility screen using Mueller Hinton plates.

FIG. 3 illustrates detecting active and inactive particles. (a) asolution of target (large circles) and non-target (small circles)particles; (b) active particle decorated with antibodies or smallpeptides; (c) decorated target particle separated from non-targetparticles and concentrated by stochastic confinement; (d) rapiddetection and optical readout.

FIG. 4 illustrates stochastic confinement of particles by (a)encapsulation in droplets, (b) placement in pores of a membrane, and (c)confinement on materials with restricted transport.

FIG. 5 is an illustrative method for analyte detection. (a) particlesstochastically confined into plugs which undergo two-stage amplificationon chip to give a macroscale readout; (b) particles confined onmembranes which translate their activity to a signal seen by the nakedeye on an upper layer; (c) particles trapped in a gel with amplificationcascades incorporated localize the output signal over the activeparticle.

FIG. 6 is an illustrative method used to identify the minimal inhibitoryconcentration (MIC) of cefoxitin (CFX) for Methicillin Sensitive S.aureus (MSSA) and Methacillin Resistant S. aureus (MRSA). (a) aschematic drawing illustrates formation of plugs of bacteria, viabilityindicator, and an antibiotic at varying concentrations; (b and c) Using24 mg/L CFX as the baseline, graphs show the average change in intensityof plugs greater than (solid) and less than (striped) 3 times thebaseline for MRSA (b) and MSSA (c).

FIG. 7 is an illustrative combination of stochastic confinement with theplug-based microfluidic assay used to determine susceptibility ofbacteria to an antibiotic in a natural matrix, blood plasma. (a) aschematic drawing illustrating formation of plugs of bacteria, viabilityindicator, antibiotic, and plasma/LB mixture; (b and c) Images andlinescans of four representative plugs made from a 1:1 blood plasma/LBsample inoculated with MRSA without (left) and with (right) the additionof AMP; (d and e) Images and linescans of four representative plugs madefrom a 1:1 blood plasma/LB sample inoculated with MSSA without (left)and with (right) the addition of AMP

FIG. 8 is an illustrative schematic description of a test strip withamplification system.

FIG. 9 is an illustrative schematic drawing of a test strip with bothdetection region and control “timer” region.

FIG. 10 is an illustrative chemical amplification process involving aone-step positive feedback.

FIG. 11 is an illustrative chemical amplification process involving atwo-step positive feedback.

FIG. 12 is an illustrative chemical amplification process with a cascadeof two positive feedback loops.

FIG. 13 is an illustrative two-step amplification cascade involvingblood coagulation enzymes.

FIG. 14 is a graph of time to get response versus amount of inputobtained by simulation.

FIG. 15 is a graph of blood clotting time varied with size of patch oftissue factor.

FIG. 16 is an illustrative example of combining amplification cascadeswith stochastic confinement.

FIG. 17 is an illustrative example of selective detection of particlesby using stochastic confinement.

DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERREDEMBODIMENTS Definitions

The term “organism” refers to any organisms or microorganism, includingbacteria, yeast, fungi, viruses, protists (protozoan, micro-algae),archaebacteria, and eukaryotes. The term “organism” refers to livingmatter and viruses comprising nucleic acid that can be detected andidentified by the methods of the invention. Organisms include, but arenot limited to, bacteria, archaea, prokaryotes, eukaryotes, viruses,protozoa, mycoplasma, fungi, and nematodes. Different organisms can bedifferent strains, different varieties, different species, differentgenera, different families, different orders, different classes,different phyla, and/or different kingdoms.

Organisms may be isolated from environmental sources including soilextracts, marine sediments, freshwater sediments, hot springs, iceshelves, extraterrestrial samples, crevices of rocks, clouds, attachedto particulates from aqueous environments, involved in symbioticrelationships with multicellular organisms. Examples of such organismsinclude, but are not limited to Streptomyces species anduncharacterized/unknown species from natural sources.

Organisms included genetically engineered organisms.

Further examples of organisms include bacterial pathogens such as:Aeromonas hydrophila and other species (spp.); Bacillus anthracis;Bacillus cereus; Botulinum neurotoxin producing species of Clostridium;Brucella abortus; Brucella melitensis; Brucella suis; Burkholderiamallei (formally Pseudomonas mallei); Burkholderia pseudomallei(formerly Pseudomonas pseudomallei); Campylobacter jejuni; Chlamydiapsittaci; Clostridium botulinum; Clostridium botulinum; Clostridiumperfringens; Coccidioides immitis; Coccidioides posadasii; Cowdriaruminantium (Heartwater); Coxiella burnetii; Enterovirulent Escherichiacoli group (EEC Group) such as Escherichia coli-enterotoxigenic (ETEC),Escherichia coli-enteropathogenic (EPEC), Escherichia coli —O157:H7enterohemorrhagic (EHEC), and Escherichia coli-enteroinvasive (EIEC);Ehrlichia spp. such as Ehrlichia chaffeensis; Francisella tularensis;Legionella pneumophilia; Liberobacter africanus; Liberobacter asiaticus;Listeria monocytogenes; miscellaneous enterics such as Klebsiella,Enterobacter, Proteus, Citrobacter, Aerobacter, Providencia, andSerratia; Mycobacterium bovis; Mycobacterium tuberculosis; Mycoplasmacapricolum; Mycoplasma mycoides ssp mycoides; Peronosclerosporaphilippinensis; Phakopsora pachyrhizi; Plesiomonas shigelloides;Ralstonia solanacearum race 3, biovar 2; Rickettsia prowazekii;Rickettsia rickettsii; Salmonella spp.; Schlerophthora rayssiae varzeae;Shigella spp.; Staphylococcus aureus; Streptococcus; Synchytriumendobioticum; Vibrio cholerae non-01; Vibrio cholerae O1; Vibrioparahaemolyticus and other Vibrios; Vibrio vulnificus; Xanthomonasoryzae; Xylella fastidiosa (citrus variegated chlorosis strain);Yersinia enterocolitica and Yersinia pseudotuberculosis; and Yersiniapestis.

Further examples of organisms include viruses such as: African horsesickness virus; African swine fever virus; Akabane virus; Avianinfluenza virus (highly pathogenic); Bhanja virus; Blue tongue virus(Exotic); Camel pox virus; Cercopithecine herpesvirus 1; Chikungunyavirus; Classical swine fever virus; Coronavirus (SARS); Crimean-Congohemorrhagic fever virus; Dengue viruses; Dugbe virus; Ebola viruses;Encephalitic viruses such as Eastern equine encephalitis virus, Japaneseencephalitis virus, Murray Valley encephalitis, and Venezuelan equineencephalitis virus; Equine morbillivirus; Flexal virus; Foot and mouthdisease virus; Germiston virus; Goat pox virus; Hantaan or other Hantaviruses; Hendra virus; Issyk-kul virus; Koutango virus; Lassa fevervirus; Louping ill virus; Lumpy skin disease virus; Lymphocyticchoriomeningitis virus; Malignant catarrhal fever virus (Exotic);Marburg virus; Mayaro virus; Menangle virus; Monkeypox virus; Mucambovirus; Newcastle disease virus (VVND); Nipah Virus; Norwalk virus group;Oropouche virus; Orungo virus; Peste Des Petits Ruminants virus; Piryvirus; Plum Pox Potyvirus; Poliovirus; Potato virus; Powassan virus;Rift Valley fever virus; Rinderpest virus; Rotavirus; Semliki Forestvirus; Sheep pox virus; South American hemorrhagic fever viruses such asFlexal, Guanarito, Junin, Machupo, and Sabia; Spondweni virus; Swinevesicular disease virus; Tick-borne encephalitis complex (flavi) virusessuch as Central European tick-borne encephalitis, Far Eastern tick-borneencephalitis, Russian spring and summer encephalitis, Kyasanur forestdisease, and Omsk hemorrhagic fever; Variola major virus (Smallpoxvirus); Variola minor virus (Alastrim); Vesicular stomatitis virus(Exotic); Wesselbron virus; West Nile virus; Yellow fever virus; andSouth American hemorrhagic fever viruses such as Junin, Machupo, Sabia,Flexal, and Guanarito.

Further examples of organisms include parasitic protozoa and worms, suchas: Acanthamoeba and other free-living amoebae; Anisakis sp. and otherrelated worms Ascaris lumbricoides and Trichuris trichiura;Cryptosporidium parvum; Cyclospora cayetanensis; Diphyllobothrium spp.;Entamoeba histolytica; Eustrongylides sp.; Giardia lamblia; Nanophyetusspp.; Shistosoma spp.; Toxoplasma gondii; Filarial nematodes andTrichinella. Further examples of analytes include allergens such asplant pollen and wheat gluten.

Further examples of organisms include fungi such as: Aspergillus spp.;Blastomyces dermatitidis; Candida; Coccidioides immitis; Coccidioidesposadasii; Cryptococcus neoformans; Histoplasma capsulatum; Maize rust;Rice blast; Rice brown spot disease; Rye blast; Sporothrix schenckii;and wheat fungus.

Further examples of organisms include worms such as C. Elegans andpathogenic worms nematodes.

“Particle” as used herein refers to an organism, molecule, cell, a viralparticle, spore, and the like.

“Patient sample” refers to a sample obtained from a patient or personand includes blood, feces, urine, saliva or other bodily fluid,preferably blood. Food samples may also be analyzed.

“Sample” refers to any sample potentially comprising an organism.Environments for finding organisms include, but are not limited togeothermal and hydrothermal fields, acidic soils, sulfotara and boilingmud pots, pools, hot-springs and geysers where the enzymes are neutralto alkaline, marine actinomycetes, metazoan, endo and ectosymbionts,tropical soil, temperate soil, arid soil, compost piles, manure piles,marine sediments, freshwater sediments, water concentrates, hypersalineand super-cooled sea ice, arctic tundra, Sargosso sea, open oceanpelagic, marine snow, microbial mats (such as whale falls, springs andhydrothermal vents), insect and nematode gut microbial communities,plant endophytes, epiphytic water samples, industrial sites and ex situenrichments. Additionally, a sample may be isolated from eukaryotes,prokaryotes, myxobacteria (epothilone), air, water, sediment, soil orrock, a plant sample, a food sample, a gut sample, a salivary sample, ablood sample, a sweat sample, a urine sample, a spinal fluid sample, atissue sample, a vaginal swab, a stool sample, an amniotic fluid sampleand/or a buccal mouthwash sample.

Microfluidics is an attractive platform for rapid single-cell functionalanalysis. (M. Y. He, J. S. Edgar, G. D. M. Jeffries, R. M. Lorenz, J. P.Shelby and D. T. Chiu, Anal. Chem., 2005, 77, 1539-1544; A. Grodrian, J.Metze, T. Henkel, K. Martin, M. Roth and J. M. Kohler, Biosens.Bioelectron., 2004, 19, 1421-1428; D. B. Weibel, W. R. DiLuzio and G. M.Whitesides, Nat. Rev. Microbiol., 2007, 5, 209-218; Y. Marcy, T. Ishoey,R. S. Lasken, T. B. Stockwell, B. P. Walenz, A. L. Halpern, K. Y.Beeson, S. M. D. Goldberg and S. R. Quake, PLoS Genet., 2007, 3,1702-1708; J. El-Ali, S. Gaudet, A. Gunther, P. K. Sorger and K. F.Jensen, Anal. Chem., 2005, 77, 3629-3636; A. Huebner, M. Srisa-Art, D.Holt, C. Abell, F. Hollfelder, A. J. Demello and J. B. Edel, Chem.Commun., 2007, 1218-1220; H. M. Yu, C. M. Alexander and D. J. Beebe, LabChip, 2007, 7, 726-730; C. J. Ingham, A. Sprenkels, J. Bomer, D.Molenaar, A. van den Berg, J. Vlieg and W. M. de Vos, Proc. Natl. Acad.Sci. U.S.A., 2007, 104, 18217-18222; R. D. Whitaker and D. R. Walt,Anal. Chem., 2007, 79, 9045-9053.) Plugs, for example, droplets ofaqueous solution surrounded by a fluorinated carrier fluid, provide asimple platform for manipulating samples with no dispersion or losses tointerfaces. (H. Song, D. L. Chen and R. F. Ismagilov, Angew. Chem.-Int.Edit., 2006, 45, 7336-7356; H. Song, J. D. Tice and R. F. Ismagilov,Angew. Chem.-Int. Edit., 2003, 42, 768-772.) Microfluidic plug-basedassays provide the ability to reduce detection time by confiningbacterium into nanoliter-sized plugs. This confinement, referred to as“stochastic confinement” decreases detection time by confining thesample into plugs that either have a single bacterium, or are empty.This approach increases the effective concentration of the bacterium,and allows released molecules to accumulate in the plug. Such stochastictrapping is commonly used for single-cell analysis in microfluidicdevices, (M. Y. He, J. S. Edgar, G. D. M. Jeffries, R. M. Lorenz, J. P.Shelby and D. T. Chiu, Anal. Chem., 2005, 77, 1539-1544; Y. Marcy, T.Ishoey, R. S. Lasken, T. B. Stockwell, B. P. Walenz, A. L. Halpern, K.Y. Beeson, S. M. D. Goldberg and S. R. Quake, PLoS Genet., 2007, 3,1702-1708; A. Huebner, M. Srisa-Art, D. Holt, C. Abell, F. Hollfelder,A. J. Demello and J. B. Edel, Chem. Commun., 2007, 1218-1220; S.Takeuchi, W. R. DiLuzio, D. B. Weibel and G. M. Whitesides, Nano Lett.,2005, 5, 1819-1823; P. Boccazzi, A. Zanzotto, N. Szita, S. Bhattacharya,K. F. Jensen and A. J. Sinskey, App. Microbio. Biotech., 2005, 68,518-532; V. V. Abhyankar and D. J. Beebe, Anal. Chem., 2007, 79,4066-4073) and similar techniques have been used for single molecule andsingle enzyme work. (H. H. Gorris, D. M. Rissin and D. R. Walt, Proc.Natl. Acad. Sci. U.S.A., 2007, 104, 17680-17685; A. Aharoni, G. Amitai,K. Bernath, S. Magdassi and D. S. Tawfik, Chem. Biol., 2005, 12,1281-1289; O. J. Miller, K. Bernath, J. J. Agresti, G. Amitai, B. T.Kelly, E. Mastrobattista, V. Taly, S. Magdassi, D. S. Tawfik and A. D.Griffiths, Nat. Methods, 2006, 3, 561-570; J. Huang and S. L. Schreiber,Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 13396-13401; D. T. Chiu, C. F.Wilson, F. Ryttsen, A. Stromberg, C. Farre, A. Karlsson, S. Nordholm, A.Gaggar, B. P. Modi, A. Moscho, R. A. Garza-Lopez, O. Orwar and R. N.Zare, Science, 1999, 283, 1892-1895; J. Yu, J. Xiao, X. J. Ren, K. Q.Lao and X. S. Xie, Science, 2006, 311, 1600-1603.) Microfluidics alsoenables simultaneous execution of numerous assays of bacterial functionfrom a single bacterial sample in the same experiment, which isespecially useful for rapid antibiotic susceptibility screening.Previously, gel microdroplets had been utilized for susceptibilityscreening. (Y. Akselband, C. Cabral, D. S. Shapiro and P. McGrath, J.Microbiol. Methods, 2005, 62, 181-197; C. Ryan, B. T. Nguyen and S. J.Sullivan, J. Clin. Microbiol., 1995, 33, 1720-1726) However, this methoddid not take advantage of the stochastic confinement effects in plugs orhigh-throughput screening methods of current microfluidic technologies.Confinement effects as described herein are increased if the geldroplets are surrounded by a barrier substantially impermeable toreleased products (for example, a fluorous liquid or a non-poroussolid). An immiscible fluid surrounding the droplet will form a barrierto prevent or reduce loss of released products from a cell, enabling thereleased products to accumulate more rapidly and reach higherconcentrations in the droplet Microfluidic technology offers twoadvantages over traditional bacterial detection and drug screeningmethods: 1) stochastic confinement of single cells from dilute samplesconcentrates the bacteria, eliminates the need for pre-incubation, andreduces detection time; 2) each assay can be performed by using anindividual bacterium, enabling hundreds of assays to be performed usinga single, low density bacterial sample without pre-incubation. Thistechnology will reduce the time needed to diagnose bacterial infectionsand enable patient-specific antibiotic regimens. This technology alsohas the advantage of separating objects into individual and separatevolumes of fluid by forming plugs. A further advantage is that by usingtechniques such as the hybrid method to perform high throughputscreening of multiple reagents and conditions using only a small volumesample.

Examples of microfluidic technology including descriptions of uses,applications and techniques for plug-based methods of analysis,manipulation of plugs, hybrid plug merging, cartridge formation, use andhandling, and holding component and loading component handling, use andformation, use of markers and fluid handling include U.S. Pat. No.7,029,091; U.S. Published Patent Applications 2005/0087122 A1,2005/0019792 A1, 2007/0172954 A1, 2007/0195127 A1, 2007/0052781 A1,2006/0003439 A1, 2006/0094119 A1, 2006/0078893 A1; 2006/0078888 A1,2007/0184489 A1, 2007/0092914 A1, 2005/0221339 A1, 2007/0003442 A1,2006/0163385 A1, 2005/0172476 A1, 2008/0003142 A1, 2008/0014589 A1; andWIPO published international applications WO 07/081,386 A2, WO07/081,387 A1, WO 07/133,710 A2, WO 07/081,385 A2, WO 08/063,227 A2, WO07089541 A2, WO 07/030,501 A2, WO 06/096571 A2. These references areincorporated by reference in their entirety.

Approaches that may be used for bacterial detection include: 1) the useof cartridges (cartridges pre-loaded with reagents for differentdetection methods/growth media/antibiotics may be used to detect andidentify bacteria); 2) the hybrid method to test manyantibiotics/substrates/detection growth conditions or culturingconditions at different concentrations; and 3) screening/sorting.

In addition to concentrating the sample by stochastic confinement, othermicrofluidic on-chip approaches may be used to preconcentrate a samplebefore detection. One method is to flow the initial sample through adevice which contains a structure (such as a filter) that would collectall of the objects (cells, particles, molecules or proteins bound toparticles) needed to be detected. The structure traps the objects(through size exclusion such as a filter or through specific chemical orphysical interactions with the objects) but enables the aqueous fluid inwhich the objects are suspended to pass through the structure. Once allof the objects have been collected in the structure, the objects may beresuspended by another aqueous flow such that the volume of aqueousfluid used to resuspend the objects is less than the volume in which theobjects were originally suspended. The resuspended objects, now at ahigher concentration, may then be loaded into plugs or droplets forfurther concentration due to stochastic confinement.

Other concentration techniques may be used such as centrifugation,attaching magnetic beads to the particles of interest, or havingsurfaces which selectively bind to the target and then release thetarget at a later time. For example, see S. Song and A Singh, Analyticaland Bioanalytical Chemistry, Volume 384, Number 1/January, 2006, 41-43,and P. Gridzinski, J Yang, R H Liu, M D Ward, Biomedical Microdevices,Volume 5, Number 4/December, 2003, pg. 303-310.

The experiments may be done by slowly flowing plugs into a tube,incubating them as they flow (with heating/cooling for PCR if need bealong the way), flowing them by the detector, and then dumping them.This may address the “storage of 1,000,000 plugs” problem.

The 1,000,000 plugs problem with stochastic confinement refers to thefact that to concentrate a sample 1,000,000 fold, 999,999 empty plugsmust be formed for every 1,000,000 fold concentrated plug. One approachto this problem is do some initial screening using a less sophisticatedmethod (such as flow cytometry or optical scanning) to sort plugs intooccupied/unoccupied groups. These simple tests would not provide adetailed characterization of the object in the plug, but would rapidlydetermine whether or not the plug is occupied by an object. Then theadvantage of stochastic confinement can still be realized by runningmore tests to rapidly characterize the occupied plugs. It is alsopossible to run an initial screen that does take advantage of stochasticconfinement, by first running a general test for the presence of theobject (a simple fluorescent viability assay for bacteria) and thensorting the plugs into occupied/unoccupied before adding additional setsof reagents to test for further characterization of the object. It isalso possible to take a blood sample (for example 10 mL), make it into abulk emulsion, apply the procedure above, and then sort the plugs oranalyze the plugs using flow cytometry and the like.

A possible method for encapsulating large numbers of cells is similar tosequential merging of reagents to plugs flowing in a 1D microfluidicchannel (straight channel). However, the cells are introduced into a 2Dchannel (width of channel much larger than width of cell), and the cellsflow through regions in which various reagents are applied to the cells.The 2D channel should be approximately similar in height to the cellssuch that the cells flowing through the device form a monolayer. Fordetection, plugs may or may not be encoded. If the plugs are notencoded, plugs may be analyzed for those that respond for more complextests like susceptibility tests. Plugs may be encoded by position, or byan internal marker (for example a fluorescent marker of differentcolors) if not encoded. The same tests may be done by performingmultiple non-encoded tests in parallel.

Blood Cultures

There are many variables involved in performing a blood culture. Beforea person's blood is drawn, the physician must make several decisionsbased on knowledge of infections and the person's clinical condition andmedical history.

Several groups of microorganisms, including bacteria, viruses, mold, andyeast, can cause blood infections. The bacteria group can be furtherbroken down into aerobes and anaerobes. Most aerobes do not need oxygento live. They can grow with oxygen (aerobic microbes) or without oxygen(anaerobic microbes).

Based on the clinical condition of the patient, the physician determineswhat group of microorganisms is likely to be causing the infection andthen orders one or more specific types of blood culture, includingaerobic, anaerobic, viral, or fungal (for yeasts and molds). Eachspecific type of culture is handled differently by the laboratory. Mostblood cultures test for both aerobic and anaerobic microbes. Fungal,viral, and mycobacterial blood cultures can also be done, but are lesscommon.

The physician must also decide how many blood cultures should be done.One culture is rarely enough, but two to three are usually adequate.Four cultures are occasionally required. Some factors influencing thisdecision are the specific microorganisms the physician expects to findbased on the person's symptoms or previous culture results, and whetheror not the person has had recent antibiotic therapy.

The time at which the cultures are to be drawn is another decision madeby the physician. During most blood infections (called intermittentbacteremia) microorganisms enter the blood at various time intervals.Blood drawn randomly may miss the microorganisms. Since microorganismsenter the blood 30-90 minutes before the person's fever spikes,collecting the culture just after the fever spike offers the bestlikelihood of finding the microorganism. The second and third culturesmay be collected at the same time, but from different places on theperson, or spaced at 30-minute or one-hour intervals, as the physicianchooses. During continuous bacteremia, such as infective endocarditis,microorganisms are always in the blood and the timing of culturecollection is less important. Blood cultures should always be collectedbefore antibiotic treatment has begun.

Bacteria are the most common microorganisms found in blood infections.Laboratory analysis of a bacterial blood culture differs slightly fromthat of a fungal culture and significantly from that of a viral culture.

Blood is drawn from a person and put directly into a blood culturebottle containing a nutritional broth. After the laboratory receives theblood culture bottle, several processes must be completed: providing anenvironment for the bacteria to grow; detecting the growth when itoccurs; identifying the bacteria that grow; testing the bacteria againstcertain antibiotics to determine which antibiotic will be effective.

Given that a typical 5 mL blood sample from a patient with bacteremiacontains a cell density of 100 CFU/mL, (L. G. Reimer, M. L. Wilson andM. P. Weinstein, Clin. Microbiol. Rev., 1997, 10, 444-7) the methods ofthe present invention are capable of performing dozens of functionaltests on such a sample. Patient-specific characterization of bacterialspecies not only allows more rapid and effective treatment, but alsoenables in-depth characterization of bacterial infections at thepopulation level. Such detailed characterization can aid in tracking andidentifying new resistance patterns in bacterial pathogens. (S. K.Fridkin, J. R. Edwards, F. C. Tenover, R. P. Gaynes and J. E. McGowan,Clin. Infect. Dis., 2001, 33, 324-329; and R. T. Horvat, N. E. Klutman,M. K. Lacy, D. Grauer and M. Wilson, J. Clin. Microbiol., 2003, 41,4611-4616) The principles of these methods, stochastic single-cellconfinement and multiple functional assays without samplepre-incubation, can also be applied to other areas, including performingfunctional tests on field samples, detecting contamination of food orwater, separating and testing samples with mixtures of species,measuring functional heterogeneity in bacterial populations, andmonitoring industrial bioprocesses.

Other patient samples that may be collected include feces, urine orsaliva. The latter would be useful for assessing oral health. Tears maybe collected for diagnosing eye infections. Interstitial fluid (alsoreferred to as tissue fluid or intercellular fluid) may be collected anduse to diagnose infections in the pleural space around the lungs.

Examples of bacterial infections, include, but are not limited to thoselisted in Table 1.

TABLE 1 Types of Bacterial Infection Type of Infection DescriptionExamples Inapparent No detectable clinical Asymptomatic (subclinical)symptoms of infection gonorrhea in women and men Dormant (latent)Carrier state Typhoid carrier Accidental Zoonosis or Anthrax,cryptococcal environmanetal or infection, and inadvertent exposureslaboratory exposure, respectively Opportunistic Infection caused bySerrati or Candida normal flora or infection of the transient bacteriagenitourinary tract when normal host defenses are compromised PrimaryClinically apparent Shigella dysentery (e.g. invasion and multiplicationof microbes in body tissues, causing local tissue injury) SecondaryMicrobial invasion Bacterial pneumonia subsequent to primary followingviral lung infection infection Mixed Two or more Anaerobic abscess (E.coli microbes infecting the and Bacteroides same tissue fragilis) AcuteRapid onset (hours or Diptheria days); brief duration (days or weeks)Chronic Prolonged duration Mycobacterial (months or years) diseases (tusand leprosy) Localized Confined to a small Staphylococcal boil area orto an organ Generalized Disseminated to many Gram-negative body regionsbacteria (gonococcernia) Pyogenic Pus-forming Staphylococcal andstreptococcal infection Retrograde Microbes ascending E. coli urinarytract in a duct or tube infection against the flow of secretions orexcretions Fulminant Infections that occur Airborne Yersinia suddenlyand pestis (pneumonic intensely plague)

Bacterial Detection

To monitor the presence and metabolically active bacteria in plugs, afluorescent viability indicator alamarBlue® was added to the cultures.The active ingredient of alamarBlue is the fluorescent redox indicatorresazurin. (J. O'Brien and F. Pognan, Toxicology, 2001, 164, 132-132.)Resazurin is reduced by electron receptors used in cellular metabolicactivity, such as NADH and FADH, to produce the fluorescent moleculeresofurin. Therefore, fluorescence intensity in a plug is correlatedwith the presence and metabolic activity of a cell, in this case, abacterium. Because resazurin indicates cell viability, resazurin-basedassays have been used previously in antibiotic testing. (S. G.Franzblau, R. S. Witzig, J. C. McLaughlin, P. Torres, G. Madico, A.Hernandez, M. T. Degnan, M. B. Cook, V. K. Quenzer, R. M. Ferguson andR. H. Gilman, J. Clin. Microbiol., 1998, 36, 362-366; A. Martin, M.Camacho, F. Portaels and J. C. Palomino, Antimicrob. Agents Chemother.,2003, 47, 3616-3619; K. T. Mountzouros and A. P. Howell, J. Clin.Microbiol., 2000, 38, 2878-2884; C. N. Baker and F. C. Tenover, J. Clin.Microbiol., 1996, 34, 2654-2659.) Resazurin may be used to detect boththe presence of a live bacterium and the response of bacteria to drugs,such as antibiotics. Stochastic confinement decreases detection timebecause in a plug that has the bacterium, the bacterium is at aneffectively higher concentration than in the starting solution, and thesignal-to-noise required for detection is reached sooner since theproduct of reduction of resazurin accumulates in the plug more rapidly.

To demonstrate the ability of stochastic confinement to reduce detectiontime, a single sample of Staphylococcus aureus (S. aureus) containingthe fluorescent viability indicator was split. Half of the culture wasused to generate plugs of nanoliter volume, and the other half remainedas a milliliter-scale culture. Both the nanoliter plugs and themilliliter-scale culture were incubated for 2.8 h at 37° C. Afterincubation, the milliliter-scale culture was used to form plugs. Thisexperimental procedure is illustrated in FIG. 1 b. Line scans indicatethat confining the bacteria at the beginning of incubation (t=0), led toa few occupied plugs with a high fluorescence intensity and many emptyplugs with low fluorescence intensity (solid line). All plugs made fromthe milliliter-scale culture had an intermediate intensity (dottedline). Confining bacteria into plugs of nanoliter volume reduced thetime required to detect a change in fluorescence intensity of theviability indicator. Bacteria confined to and incubated innanoliter-sized plugs showed a greater change in fluorescence intensityafter 2.8 h than the bacteria incubated in the “unconfined”milliliter-scale culture (FIG. 1 c). Line scans of the plugs of bacteriathat were incubated in plugs showed many empty plugs with lowfluorescence intensity and a few occupied plugs with high fluorescenceintensity (FIG. 1 b, top). However, lines scans of plugs of bacteriathat were incubated in the milliliter-scale culture have a lower,uniform fluorescence intensity (FIG. 1 b, bottom). Therefore, bacteriaconfined to nanoliter-sizes plugs may be detected earlier than bacteriain a milliliter-scale culture.

In plugs containing single bacterium, the detection time wasproportional to plug volume. Detection time was defined as the time atwhich the increase in fluorescence intensity reached a maximum. Whensingle bacterium were confined in plugs ranging from 1 mL to 1500 mL involume, detection time increased with the log of plug volume (FIG. 1 c),implying that bacteria were dividing exponentially inside the plugs.This result is similar to previous estimates that detection timedecreases by about 1.5 h for every order of magnitude increase in celldensity. (P. Kaltsas, S. Want and J. Cohen, Clin. Microbiol. Infect.,2005, 11, 109-114) The detection times measured for bacteria incubatedin plugs were similar to detection times measured for bacteria incubatedin a 96 well plate from cultures with similar initial cell densities(FIG. 1 d). This result implies that incubation in plugs had no adverseeffects on growth of bacteria.

Detecting low concentrations of species (down to single molecules andsingle bacteria) is a challenge in food, medical, and securityindustries. Plugs may allow one to concentrate such samples and performanalysis. For example, a sample containing small amounts of DNA ofinterest in the presence of an excess of other DNA may be amplified.Amplification may be detected if plugs are made small enough that someplugs contain single DNA molecules of interest, and other plugs containno DNA molecules of interest. This separation into plugs effectivelycreates plugs with higher DNA of interest concentration than in theoriginal sample. Amplification of DNA in those plugs, for example byPCR, may lead to higher signal than amplification of the originalsample. In addition, localization of bacteria in plugs by a similarmethod may create a high local concentration of bacteria (1 per verysmall plug), making them easier to detect. For some bacteria that usequorum sensing, this may be a method to activate and detect them. Suchbacteria may be inactive/non-pathogenic and difficult to detect at lowconcentrations due to lack of activity, but at a high concentration ofbacteria, the concentration of a signaling molecule increases,activating the bacteria. If a single bacterium is localized in a plug,the signaling molecule produced by a bacterium cannot diffuse away andits concentration will rapidly increase, triggering activation of thebacterium, making it possible for detection. In addition, plugs may beused to localize cells and bacteria by creating gels or matrixes insideplugs. Bacteria and other species (particles and molecules) may becollected and concentrated into plugs by putting air through a plugfluid such as water, and then using that plug fluid to generate plugs.For example, by making smaller plugs from the initial plug, some of thenewly formed smaller plugs will contain sample while other plugs willnot contain the sample, but only buffer, for example. This results inconcentrated sample containing plugs because some of the plugs do notcontain any of the sample.

This method is not limited to liquid samples. Microorganisms and otherparticulate matter can be detected in gaseous samples, such as samplesof air taken at airports or along train routes. There are numerousmethods for collecting airborne particles in water, for example, asdescribed in U.S. Pat. Nos. 7,201,878, 7,243,560, and 5,855,652 allincorporated by reference herein in their entirety. After collecting theairborne particulate matter in water, these samples can then be addeddirectly to the fluorinated oil carrier fluid to form plugs.

PCR techniques are disclosed in the following published US patentapplications and International patent applications: US 2008/0166793 A1,WO 08/069,884 A2, US 2005/0019792 A1, WO 07/081,386 A2, WO 07/081,387A1, WO 07/133,710 A2, WO 07/081,385 A2, WO 08/063,227 A2, US2007/0195127 A1, WO 07/089,541 A2, WO 07030501 A2, US 2007/0052781 A1,WO 06096571 A2, US 2006/0078893 A1, US 2006/0078888 A1, US 2007/0184489A1, US 2007/0092914 A1, US 2005/0221339 A1, US 2007/0003442 A1, US2006/0163385 A1, US 2005/0172476 A1, US 2008/0003142 A1, and US2008/0014589 A1, all of which are incorporated by reference herein intheir entirety.

Amplifications of nucleic acids have been performed via polymerase chainreactions (PCR). The key concept of PCR comprises genetic template(primer), thermostable DNA polymerase, and the circuit for regulatingtemperature. By combining these components in a miniaturizedmicrofluidic device capable of implementing cartridge and/or hybridmethods, a high concentration of interesting DNA fragments from a verysmall amount of genetic sample via trivial PCR methods may be collected.It has been confirmed that PDMS is a heat-stable material, indicating itis a suitable material for the PCR process.

With respect to the cartridge, the plug-based microfluidic platform maybe used to setup a huge number of reaction centers in nano- orpico-liter volume scale, extending into the femtoliter and microliterscales. With respect to the hybrid method, various conditions ofreactions and samples may be incorporated, split, and merged in amicrofluidic device.

For example, a sample comprising 1% of DNA of interest in the presenceof 99% of background DNA would need to be amplified enough to harvestand use. However, amplification of 1% of DNA by a factor of 100 onlyincreases the total amount of DNA by a factor of 2, resulting indetection difficulties. By making small enough plugs to comprise asingle molecule of DNA of interest, and amplifying each plug by means ofconventional PCR techniques in a microfluidic device, highly amplifiedPCR products of target 1% of DNA in a plug may be obtained. Assume thatthe probability of appearance of target DNA as a single molecule in aplug is one in every 10^(th) plug, then the ratio of target DNA/totalDNA is 1:10 in such plugs. 1000-Fold amplifications of target DNAmolecule in those plugs, affords a 100:10 ratio of target to backgroundDNA, resulting in a 10 fold increase of the total amount of DNA.Conclusively, the target DNA from the very low concentration may easilybe detected under the presence of large background signals. In themicrofluidic device, a platform cartridge incorporating a carrier fluidchannel, a sampling channel, and one or two PCR reagent channels may bemade. By regulating each fluid, individual plugs containing a singlemolecule of target DNA or different molecules of DNA from the sample maybe generated. Along the channel containing plugs, heating regions areplaced in the channel region by repeating heating and cooling processfor the denaturation and renaturation of DNA samples.

Stochastic confinement has applications in the isolation and screeningof rare particles or cells from a sample. When samples arestochastically confined, the result is a set of isolated volumes offluid, most with either no particles or 1 particle. In this way, rareparticles are segregated from ubiquitous particles. The separation ofrare particles enables the direct assay of the function and detection ofrare particles without interference from other particles in the system.

Even if a rare particle has high activity, since it is at lowconcentration in the bulk sample it may not be detectable due todilution of the signal and low signal to noise ratio as a result of lowbackground reaction from the ubiquitous particles. Once rare particlesare isolated and concentrated through stochastic confinement, other plugbased microfluidic technologies can be used to screen the rare particle.

For example, if the original sample contains 100 rare particles, sincestochastic confinement enables the separation and screening ofindividual particles, this enables the rare particle to be screenedagainst up to 100 different conditions. After confining all of theparticles into droplets, those droplets may be merged with thousands ofdifferent conditions. If the droplets are randomly merged with screeningconditions, then up to 100 different conditions will be screened againstthe rare particles. If the rare particle is allowed to divide, then theplug containing the rare particle can be split into several smallerplugs and each plug assayed or screened independently, for example,using the hybrid and/or cartridge method. Examples include combining theplug containing many rare particles with plugs of various reagents andconditions in order to determine the function and optimal conditions forthe rare particle. As an additional example, several stochasticallyconfined organisms may be allowed to grow inside an array of plugs.After the array is split into four daughter arrays, each daughter arraymay be interrogated by a different technique or reagent, whilepreserving the identity of plugs and their relationship in the daughterarray (for example, 37^(th) plug in the first array corresponds to the37^(th) plug in the second, third, and forth array). The results may becombined to provide information on the response of correspondingdaughter plugs to each of the techniques. In addition, some of thedaughter arrays may be retained as a reference culture. When the resultsfrom the other three arrays are known, the reference culture arrays maybe used for further manipulation, characterization, assaying, andisolation of organisms.

Isolation of rare particles through stochastic confinement may also becombined with the hybrid and/or cartridge method for screeninggrowth/virulence activation/assay conditions against the rare cells. Forexample, if a sample contains 10,000 cells, 100 of which belong to arare cell type, stochastic confinement may be used to isolate the 100rare cells into plugs containing only a single rare cell and no othertypes of cells. Then the plugs containing the rare cell types may beused in hybrid and/or cartridge screening by the following methods.

The plugs generated from the stochastic confinement may be combined withscreening conditions using the hybrid and/or cartridge method. Plugscontaining the rare cells are randomly distributed throughout all of theplugs generated (many of which do not contain a rare cell). If manyplugs (100's, 1000's) are merged with a single screening condition, itis likely that at least one of the plugs for that condition will containa rare cell. In this way, multiple growth/assay/virulence activationconditions may be screened against the rare cell type. A separate testmay be needed to separate plugs containing rare cells from the otherplugs generated by confinement. It may be best to sort the plugs intorare cell/common cell/empty after merging with the hybrid and/orcartridge screen to reduce detection time. Instead of sorting for rarecells first (which may take time) and then merging with screeningconditions, the screening and determining the presence of rare cell maybe done simultaneously.

Alternatively, it may be desirable to first separate out plugscontaining rare cells from plugs containing common cells or no cells.This may be done using antibodies, binding assays, testing for functionspecific to the rare cell combined with automated sorting mechanisms(optical, magnetic, FACS). Once the rare cell type plugs have beenisolated, a hybrid and/or cartridge screen can be used to screen forgrowth/assay/virulence activating conditions or to run a multitude offunctional and genetic tests on the rare cell type.

Another application of stochastic confinement is to accurately countpopulations of cells. Since confinement isolates 1 cell per plug and maybe used to perform tests to identify the type of cell in each plug,confinement may be used to determine the density (number of cells oftype X per volume of sample) or the ratio (100 cells of type X for every1 of type Y) of cells in a sample. This may also be used to find ratiosof phenotypes of cell populations that are genetically identical (25% ofStaphylococcus cells are resistant to oxacillin or 30% of cells willinduce virulence in response to host protein X).

Screening for growth conditions is an important application because anestimated 99% of all microbes cannot be cultured by standard techniques.Unculturability of these organisms may be due to: 1) nutrient levels ofmedia are too high; 2) requirement of specific ion concentrations; and3) requirement for additional factors (unusual compounds not found inmost media). Confinement has two effects: 1) it reduces competition fromother microbes, giving rare and slow growing cell types time toreproduce; and 2) it can be combined with the hybrid and/or cartridgemethod to screen for media additives and the concentration of theadditive to find new growth conditions for a previously unculturedmicrobe or a microbe which is difficult to grow or slow growing undercurrent conditions. In addition, one may also use control of surfacechemistry provided by plugs to enhance growth of organisms. Compoundsthat modulate surface chemistries may be incorporated into the hybridscreen in addition to or along with compounds modulating growthconditions.

In addition, an organism might be releasing products such as quorumsensing molecules and will not initiate growth until a thresholdconcentration of released products have accumulated. Confinement willput microbial cells at higher initial cell density and enable cells togrow and optionally activate genes associated with high cell density.Some organisms may be able to grow in culture, which were previouslybelieved to be not culturable by standard techniques due to their slowgrowth or growth to low densities. These organisms may still undergo asufficient number of divisions when stochastically confined, andtherefore allow further detection by less-sensitive techniques thatrequire multiple copies of the organism to be present. When plugs areused to create stochastic confinement, growth of the organism allowsfurther manipulation and analysis that cannot be done on a single plug(for example requiring mutually incompatible methods) by splitting theplugs, injecting reagents into them and monitoring results. These stepsmay be performed sequentially, where the results of the first experimentguides the design of the second experiment, or in parallel. Examples ofmutually incompatible methods include reagents that produce similarsignals (such as fluorescence in the same range of wavelengths), ormethods that require different conditions (such as different solvents orpH values), or require different states of the organisms (such as afunctional test that requires an alive organism, and a stainingprotocols that kills the organism).

Implementation of this type of screening may be done in many ways. Forexample, a known organism may be screened in the presence of many mediaand culturing conditions (varying ion concentrations, knownautoinducers, amount of confinement, temperature, pH, protein additives,reaction oxygen species, stress inducers; changing carbon source andconcentration of carbon source; changing nitrogen source andconcentration of nitrogen source; changing availability of various tracemetals (Mn, Mo, Cu, Pt, etc.), adding drugs known to interfere withspecific cellular activities; adding transport and ion channelinhibitors, small molecules involved in cellular communication,virulence activators, etc.). After using the hybrid method to screenthrough many conditions, functional tests or other assays may beperformed in plugs to determine if compounds have been generated withproperties of interest (such as drug targets, antibiotic compounds, ionchannel inhibitors, virulence activation, virulence inhibition,degradation of various compounds, binding affinity, etc.).

One idea to investigate molecules released by microorganisms is calledOSMAC (one strain many compounds)), as described in Big Effects fromSmall Changes: Possible Ways to Explore Nature's Chemical Diversity byHelge Bjorn Bode, Barbara Bethe, Regina Höfs, Axel Zeeck, Chem Bio ChemVolume 3 Issue 7, Pages 619-627. Small changes in culturing conditions(for example, media composition, aeration, culture vessel, addition ofenzyme inhibitors) drastically change the metabolites that are releasedfrom a cell. The molecules released may aid in detection of theorganism, or may have functional uses such as antibiotics.

Therefore, even though strain B. subtilis can be cultured and a lot isknown about its genome, useful compounds that it is capable of releasingmay be missed simply because the organism has never been grown underspecific conditions such as, for example, certain concentration ofphosphate ions, addition of protease inhibitor, and addition of 10 uMautoinducer 2. Therefore, using hybrid and/or cartridge like approaches,a larger range of metabolites, released compounds, and drug leads may beprobed simply by running high throughput screens of various mediaconditions/additives. Even changes of a single component in the media(for example, phosphate from 20 mM to 1 uM) may activate the productionand release of a previously unknown metabolite or compound.

Release of compounds is highly dependent on culturing conditions. It isknown that phosphate levels, temperature, nutrient availability allinfluence the production and release of various metabolites (J. E.González-Pastor, E. C. Hobbs, R. Losick, Science, 2003, 301, 510).Hybrid and/or cartridge methods developed previously may enable thescreening of media conditions and concentrations of additives both forcommunities, common species, and rare species of microbes.

Implementation of this type of screening may be done by taking a knownorganism and screening many media and culturing conditions (ionconcentrations, known autoinducers, amount of confinement, temperature,pH, protein additives, reaction oxygen species, stress inducers, carbonsources, concentration of carbon source, nitrogen sources, concentrationof nitrogen source, availability of various trace elements and theirchemical form (Mn, Mo, Cu, Pt, V, B etc. and corresponding ions), and/orby adding drugs known to interfere with specific cellular activities,adding transport and ion channel inhibitors, and/or small moleculesinvolved in cellular communication, virulence activators, and thelike.). After using hybrid and/or cartridge method to screen throughmany conditions, functional tests or other assays may be performed inplugs to determine if compounds have been generated with properties ofinterest (such as drug targets, antibiotic compounds, ion channelinhibitors, virulence activation, virulence inhibition, degradation ofvarious compounds, binding affinity, etc.). Alternatively, this methodmay also be used with rare cells isolated from natural samples such assoil, aquatic environments including sea water and marine sediments andsurfaces, an animal's digestive tract, environmentally contaminatedsites including soil, water or air, sludge used in environmentalremediation, etc. Cells which are unculturable (no known conditionscause them to divide/reproduce outside of natural environment) may alsobe used. If small volumes are used, activity may be detected even from asingle cell without division.

It is known that some strains of microbes will not initiate growth orsome cellular functions unless cell density is above a minimalthreshold. It is also possible that although the process is occurring,the rate at low cell densities may be so slow that it would take weeksor months to observe growth or the function. Therefore, by placingsingle cells in very confined spaces with small volumes it is likelythat they will initiate high density processes and that many processeswill have increased rates. This is especially important in the case ofrare cells, since the sample may only control 1 or a few copies of therare cell. Small volume confinement makes it possible to achieve highcell densities of rare cell types.

Stochastic confinement may be used to isolate rare organisms fromvarious sources, including: soil extract from various types of soilenvironments and soil layers (including the surface layer, subsoil,substratum), ice shelves, marine and freshwater sediments, naturallyoccurring biofilms, hot springs, hydrothermal vents, extraterrestrialsamples, crevices of rocks, attached to particulates from aqueousenvironments, growing on or inside of manmade structures, clouds,gastrointestinal tract, and found forming a symbiotic relationshipinside of a host organism. Specifically, stochastic confinement may beused to isolate rare cells from soil extract. Once rare cells have beenobtained, the plugs containing the cells may be incubated overnight toallow for growth or secretion of molecules. The plugs containing therare cells may then be used as an input into the hybrid and/or cartridgemethod. For example, to find rare cells or their secreted molecules thatmay stimulate production of antibiotics and other compounds byStreptomyces species, each plug containing the rare cell may then, forexample, be merged with 1000's of plugs containing cells of Streptomycesspecies. After plugs containing streptomyces have been merged with rarecell supernatant and incubated, screening for antibiotic production isperformed. In this way, compounds in the supernatant of the rare cell,or rare cells directly, may induce the production of new antibioticcompounds.

In another example, stochastic confinement may be used to isolate cellsfrom ocean sediments. Once plugs containing cells have been collected,the hybrid and/or cartridge method may be used to screen various mediaconditions such as phosphate concentration (from 0 to 100 μM),autoinducer 2 concentration (from 0 to 100 μM), and glucoseconcentration (from 0 to 10 mM). The cells are then incubated in the newmedia conditions. Various functional/genetic tests are performed in theplugs to determine which media conditions yield growth and or productionof compound with desired properties

Alternatively, cells which are unculturable (no known conditions causethem to divide/reproduce outside of natural environment) may be used. Ifsmall volumes are used, one may be able to detect activity even from asingle cell without division.

Plug based methods may be used to collect the lysate or cell freesupernatant from various types of cells and merge these solutions withother cells to elicit metabolite/compound production. Lysate/supernatantmay be diluted during the screen (concentration screen using hybridmethod). In this way, uncharacterized/unknown compounds/combinations ofcompounds may be screened to elicit production of useful compounds.

It should be noted that using stochastic confinement to enumerateparticles including organisms and cells would also be useful forcounting the occurrence of rare cell types in a sample. Stochasticconfinement has applications in isolation of rare particles from sampleswith many other ubiquitous particles besides a few particles ofinterest. When samples are stochastically confined, the result is a setof isolated volumes of fluid, most with either no particles or oneparticle. Rare particles are therefore separated from ubiquitousparticles. The separation of rare particles enables the direct assay ofthe rare particles without interference from other particles in thesystem. Even if a rare particle has high activity, since it is at lowconcentration in the bulk sample it may not be detectable due todilution of the signal and low signal to noise or low signal tobackground (potentially low background reaction from the ubiquitousparticles). Once rare particles are isolated and concentrated throughstochastic confinement, other plug based microfluidic technologies maybe used to screen the rare particle. If the rare particle is allowed todivide, then the plug containing the rare particle can be split intoseveral smaller plugs and assayed or screened using the hybrid and/orcartridge method (combining the plug containing many rare particles withplugs of various reagents and conditions in order to determine thefunction and optimal conditions for the rare particle). In addition, ifthe original sample contains 10 rare particles, since stochasticconfinement enables the separation and screening of individualparticles, this enables the rare particle to be screened against up to10 different conditions. When rare particles are bacteria, care must betaken with bacteria to avoid forming biofilms during incubation whichmight grow and adhere to the wall, interfering with enumerations. Thusit is preferable to use inert surfaces in plugs.

Two important purposes when dealing with the particles of interest aredetection and harvest, both of which are enabled or greatly enhanced bystochastic confinement when the particles of interest are rare particlesmixed with many other ubiquitous particles. Without stochasticconfinement, the background is too high compared to the signal fordetection, and the probability of isolating the particles of interest istoo low for harvest.

Occasions when detection is needed include, but are not limited to, whenthe particles are rare cells such as cancer cells in general, cancerstem cells, or fetal cells in maternal blood, or when the particles arebacteria or viruses causing some diseases, or when the particles aresome toxic materials released by some industrial procedure, or particlesare results of some military, civil, or natural event that needsdetection.

On the other hand, if the particles of interest have some specialfunctions that are useful, isolating and possibly multiplying them isimportant in efficiently utilizing these special functions. For example,stem cells isolated and multiplied from adults may be used fortreatment, bypassing the need for embryonic stem cells. Natural productsused in medicine may be produced by isolating and multiplying naturalcells that produce them. Bacteria with novel functions such as cleaningup hydrocarbon waste, degrading other environmental pollutants includinghalogenated compounds, converting biomass into more easily utilizablefuels such as ethanol or butanol or methane, oxidizing methane, orfixing nitrogen may be isolated, multiplied and used for appropriatepurposes.

Therefore, after the particles are separated and the plugs containingthe particles of interest are distinguished from others by a primaryassay, one or multiple further assays may be done to detect particles ofinterest and/or one or multiple types of particles detected from theprimary assay may be used alone or in combinations for appropriatefunctions.

The basis for detection of the particles include but are not limitedto: 1) surface properties including functional groups on the surface ofparticles and signaling molecules on cells (antigens, receptors, sugargroups, lipids, etc); 2) materials inside the particles (chemicalsenclosed in materials, DNA, RNA in general, microRNA, signalingmolecules in general, proteins, and the like)—these materials mayrequire further processing to the particles to be exposed and used fordetection; and 3) chemical exchange with the environment (productionand/or consumption of chemicals by material, uptake and/or secretion ofmolecules such as food, waste, signaling molecules in general, ions,novel molecules, etc. by cells). These chemical exchanges may occurnaturally or with human intervention for example by stimulation withreagents.

Specific applications

Detection of diseases by examination of fetal materials in maternalblood

Prenatal diagnosis of genetic diseases plays an important role inpregnancy, at least in informing the parents about the possibilities.Women of 35 years of age or more have high risk of abnormalities.However, since there are also many more pregnancies in the “low-risk” 26year-old group, most (about 70%) abnormalities occur in this group.(Daniilidis, A.; Kouzi-Koliakou, K., Fetal cells in maternalcirculation—potentials for prenatal control. Journal of BiologicalResearch-Thessaloniki 2006, 6, 119-130.)

Most effective current methods are invasive, where samples are takendirectly from the fetus. These procedures have a risk of miscarriage(1-2%). (Daniilidis, A.; Kouzi-Koliakou, K., Fetal cells in maternalcirculation—potentials for prenatal control. Journal of BiologicalResearch-Thessaloniki 2006, 6, 119-130.) Thus, these invasion methodsare usually applied to those in the high-risk group only. Because of therisk of miscarriage and because of the high collective occurrence ofabnormalities in the “low-risk” group that are usually not screened, anon-invasive and reliable method to detect or predict genetic disordersis in high demand.

One promising possibility is using fetal materials in maternal blood. Ifthe cells and free DNA in the mother's blood may be used effectively todetect genetic disorders, the risk of miscarriage by invasive procedureis diminished and virtually any expecting mother may have a blood drawto check for possible genetic disorders. However, the biggest challengeis the small number of fetal cells (1-6 cells/mL (Daniilidis, A.;Kouzi-Koliakou, K., Fetal cells in maternal circulation—potentials forprenatal control. Journal of Biological Research-Thessaloniki 2006, 6,119-130.)) and DNA in maternal blood.

Previously developed methods to isolate such cells involve enrichmentmethods such as density gradient centrifugation and selective lysis, andsorting methods such as fluorescence-activated cell sorting (FACS) andmagnetic-activated cell sorting (MACS). Methods to detect geneticdisorders include (PCR) and fluorescent in situ hybridization (FISH).

FACS and MACS depend on tagging the fetal cells with fluorescent orparamagnetic antibodies and using fluorescence intensity as a signal toseparate the cells with flow cytometry or using magnets to separate thecells. These techniques rely on specific antibodies. In a recent studyin which blood samples spiked with fetal nucleated red blood cells wereused to check the sorting procedure which used density-gradientcentrifugation, MACS, and selective lysis, only 37% were recovered.(Ponnusamy, S.; Mohammed, N.; Ho, S. S. Y.; Zhang, H. M.; Chan, Y. H.;Ng, Y. W.; Su, L. L.; Mahyuddin, A. P.; Venkat, A.; Chan, J.; Rauff, M.;Biswas, A.; Choolani, M., In vivo model to determine fetal-cellenrichment efficiency of novel noninvasive prenatal diagnosis methods.Prenatal Diagnosis 2008, 28, (6), 494-502.)

Prenatal diagnosis of fetal physiology, non-genetic diseases, or geneticdisorders by fetal cells and possible use of fetal cells.

Stochastic confinement and autocatalytic kinetics with threshold, asdiscussed above and in the amplification section, may allow one todetect reliably plugs containing fetal cells in maternal blood andseparate them out to use techniques in further detection or application.The two important advantages of this system versus current techniquesare:

1) Because of the threshold kinetics, the result in each plug is binaryor pseudo-binary. In other words, there is a large contrast betweenplugs containing fetal cells and other plugs (which contain other cellsor no cells). Therefore, the signal used to mechanically sort the cellsis clear and error in this step is avoided. For example, in conventionalFACS background fluorescence and photobleaching may make the resultsdeviate from being ideal.

2) Stochastic confinement allows for reliable detection even if thespecificity of an antibody label is not ideal. As long as binding to thefetal cells (such as fetal nucleated red blood cell) is at least twoorders of magnitude stronger than undesired binding to non-fetal cells,the kinetic threshold may be adjusted to lie in between the twoconcentrations of antibodies in plugs. The adjustment may be carried outby choosing an appropriate amplification method and tuning theconcentration of such method (see section about amplification). There isa need to determine properties of fetal cells (such as having aparticular disease or not). Using antibodies that selectively bind tofetal cells of interest, stochastic confinement may be used to detectsuch properties. Even though the contrast provided by the specificity ofthe antibodies may not be much, stochastic confinement and/or anamplification method may greatly enhance this contrast.

The general steps of this method include:

obtaining about 10-20 mL of blood sample from the expecting mother (thevolume is chosen because even in such cases when amplification methodsmay detect single cells, a 1 mL sample of blood still has a significantprobability to not contain the cells of interest when the concentrationof the fetal cells is 1-6 cells/mL);

coarse enrichment by density gradient centrifugation or other methods(optional);

stochastic confinement into plugs;

primary detection (such as by using antibodies (such as monoclonalantibody against H315 for trophoblasts, (Daniilidis, A.; Kouzi-Koliakou,K., Fetal cells in maternal circulation—potentials for prenatal control.Journal of Biological Research-Thessaloniki 2006, 6, 119-130) monoclonalantibody against transferring receptor for erythroblass, (Bianchi, D.W.; Flint, A. F.; Pizzimenti, M. F.; Knoll, J. H. M.; Latt, S. A.,Isolation of Fetal DNA from Nucleated Erythrocytes in Maternal Blood.Proceedings of the National Academy of Sciences of the United States ofAmerica 1990, 87, (9), 3279-3283.) etc.));

optionally if an amplification method is needed to enhance the contrast:merging with plugs containing chemicals or materials needed in theamplification method (if these chemical and materials are added with theantibody this step is not needed);

using the plugs containing fetal cells of interest for further assays(such as FISH or PCR to look for genetic disorders, or any otherpossible methods to detect certain properties of interest of fetalcells) if necessary; and/or

using the plugs containing fetal cells of interest for applications ifthere are functions associated with such fetal cells in need, with orwithout multiplying these cells.

Fetal materials (including DNA) in maternal blood and markers ofdisorder after stochastic confinement may also be detected with methodsdescribed in the section entitled “amplification.”

Detection

The following articles, describing methods for concentrating cellsand/or chemicals by making small volume plugs with low numbers of itemsto no items being incorporated into the plugs, with specificapplications involving PCR, are incorporated by reference herein: AnalChem. 2003 Sep. 1; 75(17):4591-8. Integrating polymerase chain reaction,valving, and electrophoresis in a plastic device for bacterialdetection. Koh C G, Tan W, Zhao M Q, Ricco A J, Fan Z H; Lab Chip. 2005April; 5(4):416-20. Epub 2005 Jan. 28. Parallel nanoliter detection ofcancer markers using polymer microchips. Gulliksen A, Solli L A, Drese KS, Sörensen O, Karlsen F, Rogne H, Hovig E, Sirevåg R.; Ann N Y Acad.Sci. 2007 March; 1098:375-88. Development of a microfluidic device fordetection of pathogens in oral samples using upconverting phosphortechnology (UPT). Abrams W R, Barber C A, McCann K, Tong G, Chen Z, MaukM G, Wang J, Volkov A, Bourdelle P, Corstjens P L, Zuiderwijk M, KardosK, Li S, Tanke H J, Sam Niedbala R, Malamud D, Bau H; Sensors, 2004.Proceedings of IEEE 24-27 Oct. 2004 Page(s):1191-1194 vol. 3. Amicrochip-based DNA purification and real-time PCR biosensor forbacterial detection. Cady, N.C.; Stelick, S.; Kunnavakkam, M. V.; YuxinLiu; Batt, C. A.; Science. 2006 Dec. 1; 314(5804):1464-7. MicrofluidicDigital PCR Enables Multigene Analysis of Individual EnvironmentalBacteria. Elizabeth A. Ottesen, Jong Wook Hong, Stephen R. Quake, JaredR. Leadbetter; Electrophoresis 2006, 27, 3753-3763. Automated screeningusing microfluidic chip-based PCR and product detection to assess riskof BK virus-associated nephropathy in renal transplant recipients.Govind V. Kaigala, Ryan J. Huskins, Jutta Preiksaitis, Xiao-Li Pang,Linda M. Pilarski, Christopher J. Backhouse; Journal of MicrobiologicalMethods 62 (2005) 317-326. An insulator-based (electrodeless)dielectrophoretic concentrator for microbes in water. Blanca H.Lapizco-Encinas, Rafael V. Davalos, Blake A. Simmons, Eric B. Cummings,Yolanda Fintschenko; Anal. Chem. 2004, 76, 6908-6914. ElectrokineticBioprocessor for Concentrating Cells and Molecules. Pak Kin Wong,Che-Yang Chen, Tza-Huei Wang, and Chih-Ming Ho; Lab Chip, 2002, 2,179-187. High sensitivity PCR assay in plastic micro reactors. JianingYang, Yingjie Liu, Cory B. Rauch, Randall L. Stevens, Robin H. Liu, RalfLenigk and Piotr Grodzinski; Anal. Chem. 2005, 77, 1330-1337.High-Throughput Nanoliter Sample Introduction Microfluidic Chip-BasedFlow Injection Analysis System with Gravity-Driven Flows. Wen-Bin Du,Qun Fang, Qiao-Hong He, and Zhao-Lun Fang; Science Vol 315 5 Jan. 2007,81-84. Counting Low-Copy Number Proteins in a Single Cell. Bo Huang,Hongkai Wu, Devaki Bhaya, Arthur Grossman, Sebastien Granier, Brian K.Kobilka, Richard N. Zare; Nature Biotechnology Vol 22 (4), April 2004. Ananoliter-scale nucleic acid processor with parallel architecture. HongJ W, Studer V, Hang G, Anderson W F, and Quake S R; Electrophoresis2002, 23, 1531-1536. A nanoliter rotary device for polymerase chainreaction. Jian Liu, Markus Enzelberger, and Stephen Quake; Biosensorsand Bioelectronics 20 (2005) 1482-1490. Microchamber array based DNAquantification and specific sequence detection from a single copy viaPCR in nanoliter volumes. Yasutaka Matsubara, Kagan Kerman, MasaakiKobayashi, Shouhei Yamamura, Yasutaka Morita, Eiichi Tamiya; US PatentApplication 2005/0019792 A1, “Microfluidic device and methods of usingsame”; and Nature Methods 3, 541-543 (2006) “Overview: methods andapplications for droplet compartmentalization of biology” John H Leamon,Darren R Link, Michael Egholm & Jonathan M Rothberg.

Plugs offer advantages over cuvettes. For example, with plugs, theoptical path may be made very thin, so if a bacterium is labeled (forexample, with fluorescent antibodies) it is easy to detect.Alternatively, the same effect may be obtained by squeezing a bloodsample between two coverslips separated by a 20 μm gap, which may becreated by placing a thin metal (gold or Pt) wire between the coverslips. Thus, the cover slips may be covered with something to whichbacteria stick, and factors that makes them grow biofilms or multiplyaggressively. Probes may be added for detecting these activities orcolonies.

Another advantage is diffusion control in a plug versus a coverslip orcuvette. For example, in a cuvette, a material produced by bacteria candiffuse away and become diluted. In a plug, the material produced bybacteria builds up to a high enough concentration that it is easy todetect. The material produced or the multiplying bacteria may bedetected. Once the bacteria start growing, they grow until nutrients inthe plug are depleted. Monitoring the decrease in a nutrientattributable to the presence of the bacteria provides evidence of thebacteria. For example, the decrease in O₂ attributable to the presenceof the bacteria may be done using agglutination beads.

Oxygen or other gases or mixtures of gases may need to be provided toencourage growth. Gases may be introduced by various methods includingby dissolving them in the fluorocarbon carrier fluid. Care may be takento avoid evaporation of oil/media particularly for incubation at 37° C.The plugs may be sealed in glass or placed in a Teflon capillary that ispermeable to gases. The capillary is then placed in a vial of media thatis the same (isotonic but not comprising indicators, cells and othercomponents which will not partition across tubing). In short, the oxygenconcentration of blood in any sepsis assay may need to be controlled ina way other than what is typically done with blood tests.

Many bacteria and bacterial components can directly activate individualcoagulation factors. However, direct initiation of the coagulationcascade and the formation of a propagating clot is not typicallyobserved when humans are infected. These bacterial components usuallyactivate low levels of coagulation factors, but this activation does notresult in the amplification and positive feedback necessary to form aclot that can grow and propagate. For example, Staphylococcus aureus (S.aureus) produces coagulase, a protein that binds prothrombinstoichiometrically and leads to cleavage of fibrinogen to fibrin.However, this conversion simply precipitates fibrin and does not resultin production of thrombin, feedback, or amplification of the coagulationcascade. Escherichia coli (E. coli) that express the protein Curli arealso known to activate coagulation factors, such as factor XII. Thisprocess was shown to cause slower initiation of coagulation due todepletion of factor XII. Bacteria do initiate coagulation in someorganisms, such as horseshoe crabs, but this mechanism of controllinginfection is believed to have been lost during evolution of vertebrates.

Crab blood is known to clot rapidly on contact with bacteria. Plugs withgrown bacteria may be merged with plugs that contain crab blood.Clotting occurs rapidly when the crab blood contacts the bacteria, andsecondary indicators such as fluorescent indicators or dyes for “crabthrombin” may be used to detect bacteria. Other clotting systems aredisclosed in Kastrup et al. Acc Chem. Res. “Using chemistry andmicrofluidics to understand the spatial dynamics of complex biologicalnetworks.” 2008 April; 41(4):549-58.

Bacteria export materials out of the cell, for various reasons includingto attack the host, to digest food, to fight, to signal, etc. Thesechemical messengers become more concentrated inside a plug than insidethe original sample of blood. If the chemical messenger is an enzyme, afluorogenic substrate for the enzyme would indicate the presence of thebacteria. Human blood has esterases, etc, that may interfere, but theuse of enzymes specific to bacteria would allow detection of thebacteria. In fact, if one had a panel of 30 substrates, one may set up30 tubes with 1000 plugs each, and looks for specific substrateslighting up (if there is substrate for one bacterium), or one may lookfor patterns of substrates lighting up if there is a more complicatedrelationship (each bacterium has a pattern of substrates associated withit).

Another type of molecule exported by bacteria are signaling molecules.If a single bacterium produces activators, these activators canaccumulate in the plug, turning a single bacterium ON into the attackmode, and making it easier to detect.

Many types of cells react to high cell density by activating behaviorsand specific genes through the process of quorum sensing. For instance,the human opportunistic pathogen Pseudomonas aeruginosa releasessignaling molecules (homo serine lactones or other signals known asautoinducers) which accumulate in the space surrounding the cells andenable the cells to measure their local cell density. Many pathogens(such as Pseudomonas aeruginosa) activate virulence behaviors inresponse to the activation of quorum sensing. Because detection of cellsin a virulent state is of interest, stochastic confinement would placecells at a high enough density that they should activate virulencemechanisms which may then be detected. In this way, stochasticconfinement can detect cells with the potential for virulence, even ifthey currently have not yet activated virulence in the patient.Additionally, virulence activation often involves the upregulation ofenzymes involved in infection, such as lipases, coagulases, andproteases that may be used as detection targets, specifically asdetection targets of virulent species. The virulence enzymes and otherreleased molecules may be used to activate detection mechanisms.

Another possibility is to lyse the cells in the media to detectsomething that is only produced by bacteria.

PCR may also be applied to plugs. PCR may be overwhelmed by thebackground DNA of human cells. In the case of plugs, if plugs are madesmall enough there would be only one cell per plug (human or bacterial)which eliminate background issues (if bacterial-specific primers areused background from other cells would not interfere). Binding ofbacteria to human cells, or bacteria hiding inside of human cells wouldnot be problematic due to the minimal background. Plugs may also bespiked to stimulate bacteria to make them easier to detect.

If bacteria can be detected in people at low concentration (beforeinfection becomes a real threat), these methods may be used for generalclinical practice to screen high risk patients including babies and theelderly for bacteria. Viral infections may also be monitored by thismethod by using vesicles that a virus would try to enter, and detectingthe entry by fluorogenic substrates, or by the destruction of vesicles(detected, for example, by a Ca/Fluo4 system).

Methods of detecting bacteria using microfluidic based techniquescomprise: 1) screening a sample against various reagents which resultsin a detectable signal in the presence of bacteria; and 2) screeningthese samples in parallel or in series against different drugs todetermine which drug is best suited for killing the bacteria in thesample. Part 1) of this technique may include screening a sample againstpreloaded, plug-based cartridges that comprise bacteria-specificreagents. If the sample comprises bacteria that are specific for areagent in one of the plugs a signal will be detected in that plug.Examples of the detection methods include, but are not limited to: i)magnetic based detection, ii) optical detection, and iii) oxygendetection. Part 2) of this technique may include preloading plug-basedcartridges with various antibiotics that are known to kill certain typesof bacteria. WO 05-056826 A1 which discloses methods is incorporated byreference herein in its entirety.

In some aspects of the present invention, the method of detectingbacteria comprises confinement of a bacterium into a small volume plug,wherein the confinement induces virulence activation of the bacteriathrough a quorum sensing type mechanism. The activation of virulence inthe bacteria may induce the upregulation of various virulence factorssuch as proteins, enzymes, and small molecules. The upregulation andrelease of these virulence factors may be used as a detection target forthe presence of virulent bacteria, may be used to detect a bacteriumwith the potential to activate virulence mechanisms, or may be used astargets to detect a specific species or type of bacteria. Theupregulation and release of these virulence factors such as lipases,proteases, and coagulases may be incorporated as the initial step in anenzymatic detection cascade.

In some aspects of the method of detection of bacteria, the plugcomprises a substance capable of lysing the bacteria. Lysis may beaccomplished by standard detergent-based bacterial cell lysis. Forexample, frozen and thawed cell pellets are incubated with lysis bufferthat is supplemented with lysozyme, which help disrupt cell walls(Lysozyme hydrolyzes β(1→4) linkages between N-acetylmuramic acid andN-acetyl-D-glucosamine residues in peptidoglycan and betweenN-acetyl-D-glucosamine residues in chitodextrin). Gram-negative bacteriamay be hydrolyzed in the presence of EDTA that chelates metal ions inthe outer bacterial membrane. Cells are incubated with lysis buffer forabout 30 min, on ice. If the target is a nucleic acid, proteinase shouldbe supplemented, whereas if the target is a protein, nucleases should besupplemented. To separate cell debris and insoluble protein (e.g.,inclusion bodies), the sample is centrifuged (14,000 g, 30 min, 4° C.)and the supernatant collected. The supernatant comprises the solubleprotein fraction, which can be further purified or directly analyzed,for example by SDS-PAGE.

Methods for Detecting Bacteria

In some aspects, the method for detecting bacteria comprises detectingbacteria in various samples using microfluidic based techniques, andscreening bacteria using a hybrid and/or cartridge-based method todetect microorganisms by detecting their binding to beads.

Binding bacteria to magnetic beads may be accomplished by using specific(antibodies, chemical link between bacteria and bead) or non-specific(charge of bacteria, general molecule expressed on outside of bacteria)methods. Bound and unbound magnetic beads may be separated bymicrofluidic sorting techniques which rely on differences in diffusionbased on size or changes of magnetic potential due to bound bacteria.Bound and unbound magnetic beads may also be separated using a magneticfield because migration in the field will be reduced for bound beadswith increased drag. This method may be used to isolate specific typesof bacteria from a sample or to remove all bacteria from other parts ofthe sample matrix. Liquid containing bacteria bound to beads may then beused to make plugs. Various techniques may be used to detect thepresence of a magnetic bead in a plug, including measuring the electriccurrent induced by a moving magnetic particle. Another detection methodincorporates a hard-drive head to detect magnetic particles. Anotherdetection method takes a relative measurement of the magnetic beads tomeasure relative orientation rate in the field. Another detection methodrelies on bacteria binding to multiple beads and the detection methodbeing able to distinguish between single unbound beads and groups ofbound beads (for example by changes in the amplitude of the spike in thedetector).

Magnetic beads may also be used to detect proteins or other molecules byeither chemically linking the target to the bead, or by first attachingan antibody to the target. The antibody then recognizes and attaches tothe magnetic bead.

Optical detection schemes may also be used. Beads may have an opticalsignal, and a detection method which differentiates single unbound beadsvs. groups of beads may be used to detect the target.

If the beads-based method provides means to count the number of detectedobjects, then bead detection schemes may be used to monitor theproliferation of bound objects after exposure to a compound, such as anantibiotic. The bead method may be used to isolate the bacteria from thesample and then introduce the antibiotic to the bacteria. Addition ofmore beads and enumeration of bound beads after incubation with theantibiotic may be used to determine whether or not the antibioticinhibited proliferation of the bound bacteria.

Other detection schemes may be used to detect the bacteria themselves,such as oxygen or carbon dioxide detectors. Oxygen detection schemesinclude formation of Prussian blue as a function of oxygen presence, andfluorescence based oxygen sensors.

The sample may be detected attached to a bead, free in solution, orafter deposition on the wall of the channel. Instead of a bead, bacteriamay be agglutinated.

In some aspects, an array of pre-formed nanoliter sized droplets, orplugs is generated. Each plug comprises one or more beads. In each plug,all beads are substantially similar. Each bead has a specific bindingaffinity, provided by an antibody for example, for a microorganism or asubset of microorganisms, for example, bacteria, viruses, or fungi. Thebinding affinity can also be for a specific small particle, such as apollen grain or a spore. The beads themselves are detectable, forexample a magnetic bead or a fluorescent bead. Ideally, the bindingevent is also detectable.

If the binding event of one or more beads with a bacterium is directlydetectable, the assay may be performed by the steps of injecting thesample into each of the plugs, incubating to allow the detection, andperforming the detection. Direct detection may be accomplished by takingadvantage of the difference in the magnitude and frequency of thesignals produced by many unbound beads inside of a plug vs amicroorganism that carries with it the same number of beads bound. Manyunbound beads would generate several smaller signals, while an organismwith the same number of beads bound would generate a single signal ofhigher amplitude.

If the binding event is not detectable, then the detection can beperformed by separating bound beads from unbound beads, and detectingbound beads. The separation may be performed by a range of methodsincluding a diffusive filter, a Brownian ratchet, or in the case ofmagnetic beads, a magnetic filter that applies a magnetic field to biasmotion of the beads. Separation may require separating the plug fluidfrom the carrier fluid, flowing through the filter, and re-forming theplugs by adding carrier fluid to the plug fluid that passed the filterand contains predominantly bound beads. Detection is performed by arange of methods, including scanning the detector over the array ofplugs, or flowing the array of plugs past the detector. Magneticdetection may be performed by detecting currents generated by movingmagnetic beads, and may also incorporate technologies used in hard diskdrives.

Spacer and index plugs may be used in the original array of plugs asdisclosed in WO 08-079274 A1, the entirety of which is herebyincorporated by reference. Index plugs may contain markers detectable bythe same method used to detect binding of bacteria to beads.

This method is discussed in the context of microorganisms and particles,but it is applicable to the detection of molecules and other objects byusing a larger bead carrying antibodies against the molecule, and a setof detectable beads carrying another antibody against the same molecule.In the absence of the molecule, the detectable beads do not bind largerbeads and remain dispersed. In the presence of the molecule, thedetectable beads bind to the molecules and therefore to the largerbeads, and become detectable by the methods described here.

To perform an antibiotic screen, plugs may contain antibiotics, and theplugs injected with the sample may be allowed to incubate to permitgrowth of microorganisms. Antibiotics are recognized and are substanceswhich inhibit the growth of or kill microorganisms. Examples ofantibiotics include, but are not limited to, chlorotetracycline,bacitracin, nystatin, streptomycin, polymicin, gramicidin,oxytetracyclin, chloramphenicol, rifampicin, cefsulodin, cefotiam,mefoxin, penicillin, tetracycline, chloramphenicol, minocycline,doxycycline, vancomycin, bacitracin, kanamycin, neomycin, gentamycin,erythromycin, cephalosporins, geldanamycin, and analogs thereof.Examples of cephalosporins include cephalothin, cephapirin, cefazolin,cephalexin, cephradine, cefadroxil, cefamandole, cefoxitin, cefaclor,cefuroxime, cefonicid, ceforanide, cefotaxime, moxalactam, ceftizoxime,ceftriaxone, and cefoperazone. Additional examples of antibiotics thatmay be used are in US 2007/0093894 A1, hereby incorporated by referencein its entirety. Detection of differences in growth and microbialpopulations in the absence and presence of each antibiotic would provideinformation on antibiotic susceptibility. First the bacteria in thesample are counted. Then, the bacteria sample is merged into thecartridge containing plugs of different growth media and differentantibodies along with some as “blank” media and “blank” antibioticsplugs. Recount is preformed by merging with magnetic beads to see whichone had bacteria reproducing.

In some aspects, the detectable signal is produced by the growth of thebacteria. Optical detection comprising optical methods for detection mayuse fluorescent nanoparticles instead of magnetic ones. One may also usecontrol of surface chemistry provided by plugs to enhance growth oforganisms. One method comprises merging the sample with a cartridge, thecartridge may optionally have growth tablets; monitoring bacterialgrowth by a change in oxygen concentration; and recording the readout.The readout may be but is not limited to a change in volume of oxygenbubbles the device, a change in optical signal due to the presence of anoxygen sensitive substrate (the substrate may be incorporated in manyways including in solution, on beads, or immobilized on a film thatcoats the device), or calorimetric reactions for detecting oxygen.

Bacteria detection by using agglutination may also be employed. Thismethod comprises merging sample with plugs that comprise substance thatinduces agglutination in the presence of bacteria including for example,antibody labeled beads; forming agglutination screens on a large scalefor bacteria detection; filling preformed plugs with beads covered withantibodies specific for different bacteria; and monitoring which plugsresult in clumps of beads indicating presence of bacteria. Monitoringmay be done by eye or some other detection technique. Secondaryagglutination may also be used.

In addition, multiple assays may be performed in one device, for exampleby splitting a sample into a plurality of samples using techniquesdescribed above for splitting plugs.

One strategy is to change the hydrophobicity of the channel. Aqueousplugs with fluorinated oil as the carrier fluid will not wet hydrophobicchannel walls. Therefore, to have a plug stick to the channel wall it isnecessary to create a region of the channel in which the surfacechemistry of the channel is hydrophilic.

A hydrophilic patch may be useful in 2 ways: i) a hydrophilic patch maycapture a plug and hold it in place; and ii) a hydrophilic patch maytemporarily come into contact with a plug as it is flowing by. Thisbrief contact may result in a small portion of the plug fluid beingdeposited on the hydrophilic region of the channel wall. Capturing aplug (i) or depositing a portion of a plug onto the channel wall (i) mayenable the assays to be run on the plug fluid.

Methods which require a surface such as ELISA, or an oxygen sensorincorporated into the surface, etc. may then be performed at thehydrophilic patch of channel wall. There are some papers which suggestthat measurements performed on surfaces are more sensitive thanmeasurements in bulk fluid. More generally, the concept of controllingchannel wall surface chemistry can be implemented in the trafficking andmeasurement of plug fluids.

Another strategy for manipulating plugs is to control pressure drops inthe channels by channel constriction. From Hagen-Poiseuille's law, if achannel gets narrower, the pressure drop quickly increases. If thepressure drop in one branch of a microfluidic network becomes narrow,plugs will not be able squeeze through the constriction because thecapillary pressure required for the plug to squeeze through will behigher than the pressure drop over the device. In this way, channelconstriction may be used to collect plugs and stop them from moving.

By designing a device with specified pressure drops in each channel in anetwork of branching channels, it may be possible to automatically sortthe large number of plugs needed for some stochastic confinementapplications. For example, a main channel carrying plugs splits into 10different smaller channels. If each channel has a differentdiameter/length (i.e. pressure drops for each channel are different)this would create a bias in the loading of plugs. A system may bedesigned such that the 1st 100 plugs would be loaded into channel 1,then the pressure drop in channel 1 becomes greater than channel 2 dueto the presence of many plugs, therefore the next 100 plugs would flowinto channel 2, etc. This type of bulk sorting of plugs can be achievedsimply by designing the channels with specific pressure drops and doesnot rely on turning on and off pumps, opening and closing valves, orother “active” plug sorting mechanisms.

Certain embodiments of the invention may be used to detect sepsis in 3to 4 hours from a blood sample from a patient, and in other embodimentsdetection times may be reduced to 20 minutes or less. In a 5 to 10 mLblood sample from an infected patient, there might be 100 to 1,000bacteria. Allowing the sample to culture overnight can increase thosenumbers by 10 to 100-fold. It would also be useful to know patterns ofantibiotic resistance, and current methods are very tedious.

Possible means for detecting bacteria in a sample include stains anddyes, as used in flow cytometry, and which are well known in the art.Alternatively, one may look for a uniform change in color, appearance,scattering or optical density across a plug. almarBLUE™ (resazurin) is afluorescent redox sensitive dye that can be used to detect living cells.

Certain embodiments of the invention may be used to detect differentstrains of bacteria, including Pseudomonas, Staphylococcus, E. Coli,etc.

When testing whole blood for bacteria one can use known methods to getrid of white blood cells, which would also be metabolically active,before testing for the presence of bacteria. For example, there arelysing agents well known in the art that selectively lyse eukaryoticcells.

A severely infected patient can have 10⁶ bacteria (CFU)/mL in the blood.There are known methods for detecting 10² to 10³ bacteria/mL, but onlywith culturing overnight. For example, the PCR-based LightCycler™ candetect 10³, but only can only detect bacteria with known, predetermined,target gene sequences, and it does not give any functional information,for example concerning antibiotic resistance, and it is relatively slow,taking 6 to 8 hours. Other methods, using 96 well plates havedemonstrated the detection of 10⁶ bacteria in a 200 μl sample in 2 to 3hours, 10⁵ bacteria in approximately two hours, and 10³ bacteria in 8 to9 hours. However, it is desirable to be able to detect lowconcentrations of clinically relevant bacteria in 3 to 4 hours or less.

Another means of detecting and typing cells is PCR amplification of16S-23S rRNA., as described in Vliegen, I., et al., “Rapididentification of bacteria by real-time amplification and sequencing ofthe 16S rRNA gene” Journal of Microbiological Methods 66 (2006) 156-164,and patent application WO 96/119585, hereby incorporated by reference inits entirety. This can be accelerated by using a rapid microchip PCRmethod described recently that uses infrared light to achieve a 12minute PCR reaction. See “On-chip pressure injection for integration ofinfrared-mediated DNA amplification with electrophoretic separation”Christopher J. Easley, a James M. Karlinseya and James P. Landers, LabChip, 2006, 6, 601-610.

Fluorogenic media, which change color in the presence of specificbacteria, can also be used to detect cells. Chromogenic media include,for example, Difco mEl agar, Merck/EMD Chromocult™ Coliform Agars,Chromocult™ Enterococci Agar/Broth, or Fluorocult® LMX Broth, BL mlagar, IDEXX Colilert, CPI ColiTag and Merck/EMD ReadyCult®. Typicalenzyme substrates linked to chromogens or fluorogens include ONPG, CPRG,and MUG. These are also available in ready-to-use format, e.g. BBL mlagar and ‘convenience’ packs, e.g. IDEXX Colilert, CPI ColiTag andMerck/EMD ReadyCult®.

Bacteria can also be detected using simple growth and densitymeasurements of plugs. Such measurements may aid detection andcharacterization of specific antibiotics that block the ability of thebacteria to grow, after combining plugs with specific antibiotics.

Microchannel PCR is described in U.S. Pat. No. 6,990,290, rapidbacterial PCR is described in U.S. Pat. No. 6,673,578, and a sepsisdetection chip is described in US 2005-130185 A1, all incorporated byreference herein in their entirety.

Confining bacteria in small spaces might influence their phenotype, andgene regulation. For example, one bacterium inside of a small volume mayrespond as if it is in a culture with high cell density becausecommunication molecules that it secretes, such as homoserine lactones,activate quorum sensing. This can be used to an advantage of this todecrease detection times or limits, and/or trigger virulence.

Staining

Typically to monitor bacterial growth, blood sample bottles are placedin an incubator and kept at body temperature. They are watched daily forsigns of growth, including cloudiness or a color change in the broth,gas bubbles, or clumps of bacteria. When there is evidence of growth,the laboratory does a gram stain and a subculture. To do the gram stain,a drop of blood is removed from the bottle and placed on a microscopeslide. The blood is allowed to dry and then is stained with purple andred stains and examined under the microscope. If bacteria are seen, thecolor of stain they picked up (purple or red), their shape (such asround or rectangular), and their size provide valuable clues as to whattype of microorganism they are and what antibiotics might work best. Todo the subculture, a drop of blood is placed on a culture plate, spreadover the surface, and placed in an incubator.

Aggregation of a signal can be used to detect the presence of a singleorganism in a plug. With reference to FIG. 3, a single bacterium cansimultaneously bind to many antibodies. If each of these antibodies hasa signal, then the signal would be localized and therefore becomedetectable. For instance if each antibody was tagged with a fluorescentmarker, then although single fluorescent markers may not be detected,the co-localized signal from many markers would be above the thresholdfor detection. Similarly, if each antibody was tagged with an enzyme,then many antibodies close together would create a high localconcentration of the enzyme. Many enzymatic cascades (such as initiationof blood coagulation) require a threshold local concentration of enzyme.In this way, single antibodies would not be detected, but a localcluster of antibodies all bound to the same bacteria would create adetectable local concentration of enzymes. In a more specific example,if the bacteria sample was in blood or in a solution that contained thecoagulation cascade, a detection method may involve antibodies whichbind to the bacteria but also are tagged with the metalloprotease InhA(expressed by Bacillus species which induce blood clotting). A bacteriumthat is recognized by the antibody will bind to many antibodies andcreate a cluster of antibodies and therefore a localized cluster ofInhA. The coagulation cascade will respond to the local cluster of InhAand initiate coagulation.

An additional effect of aggregation of a signal in the decrease in thebackground signal. For instance, if there are 100 molecules of signal ina 1 mL sample, then before clustering the background is 100molecules/mL. After clustering 80 of the molecules together followed bystochastic confinement (assume in a 1 femtoliter space), the signal isnow 80 molecules/fL and the background is now 20 molecules/mL because 80molecules of signal have been removed from the bulk solution. In thisway, aggregation of a signal increases the signal to noise ratio.

Another advantage of stochastic confinement is to segregate thesignal/analyte from background material. For example, in the detectionof bacterial strain “A” in a sample that contained numerous contaminantstrain “C”, assays in the bulk may experience interference from thepresence of strain “C”. This interference may be in the form of “C”influencing the behavior of “A”, or “C” influencing the components ofthe assay itself. Interference problems may be increased if the samplecontains an abundance of “C” and few “A”. By forming plugs whichseparate species “A” from “C”, the assay may be run successfully for thepresence of “A” as some plugs will contain only strain “A” and none ofstrain “C”. As long as the threshold is tuned properly, theamplification cascade can selectively respond to only the active, targetparticles even in the presence of a large excess of interferingparticles.

In addition to stochastic confinement using plug-based microfluidics,there are other methods that may be used to achieve confinement ofindividual organisms or molecules as shown in FIG. 4. One such method isto generate an array of microwells. Microwells can be defined as a smallcompartment with volumes of nanoliters or less. The well is open on thetop and the walls are impermeable to either the particles to bedetected, molecules used in the detection system, or both. Once thewells are loaded, a top barrier may be placed over the wells in order tocompletely confine the particle(s) and molecules in the well. Evenwithout complete confinement, a well with a single open wall will stillexperience some confinement effects due to decreased flux of particlesand molecules into and out of the well. Another confinement method is totrap the particles in the matrix of a gel or polymer. Confinementeffects will be achieved by reducing the diffusion of both the particlesand the molecules used in the detection scheme. In this way, high localconcentrations of molecules used for detection will accumulate aroundthe particle. Adding an impermeable boundary to the bottom and/or top ofthe matrix (such as glass, plastic, etc.) will further increaseconfinement effects due to decreased flux of the accumulating signalaway from the particle.

Stochastic confinement of individual bacteria into plugs of nanolitervolume or smaller volumes reduces detection time.

To reduce the time required to detect bacteria in a sample, amicrofluidic device was designed to confine single bacterium into plugsof nanoliter volume. In principle, when generating plugs with a smallvolume from a solution with a low concentration of bacteria, much of thevolume of the initial solution forms plugs that contain no bacteria.There are a few occupied plugs, each occupied by single bacterium. As aresult, the concentration of bacteria in the occupied plugs is greaterthan the concentration of bacteria in the initial solution. For example,if plugs of nanoliter volume were made from a culture with an initialbacterial concentration of 10⁵ CFU/mL, one in ten plugs would receive asingle bacterium, as illustrated in FIG. 1 a. The concentration of cellsin these occupied plugs would be one bacterium per nanoliter or 10⁶CFU/mL. In other words, 10⁵ CFU/mL corresponds on average to 0.1bacterium per 1 mL, and confining this solution into nanoliter plugscreates plugs with 0 bacteria per nanoliter plug and with 1 bacteriumper nanoliter plug. CFU/mL refers to the colony forming units (CFU), ameasure of live bacteria, per milliliter. Confinement effects areincreased as the volume of plugs is decreased, therefore whenconfinement effects could be achieved in nanoliter plugs, picoliterplugs or femtoliter plugs would have increased confinement effects.

Targeting virulence factor for screening antibacterial drugs is apotential way to develop novel drugs. To escape cross-resistance ofcurrent drugs, targeting to virulence factors involved in humanpathogenesis is considered essential. It has also been suggested thatusing surrogate infection model systems to screen novel drugs is a keyissue for detecting and investigating the virulence factor.

Bacterial virulence may also be used to identify new therapeutics.Targeting virulence helps to preserve the many symbioses between themicroorganism and host that contribute to human health. Targetingbacterial adhesion and toxin production and function are good approachesto developing the antivirulence drugs which will prevent the assembly ofadhesive machinery or toxin expression or secretion. Quorum-sensingsystems, two-component response systems, and biofilm formation canadditional targets for virulence factors control.

The following methods are the ways in which pathogens cause disease inhumans: adhesion, colonization, invasion, immune response inhibitors,and toxins. Since pathogenic bacteria have different methods of inducingvirulence, conditions for inducing/monitoring/evaluating virulence areuseful targets for drug discovery.

A representative example of inducing virulence is the generation ofreactive oxygen species (ROS). Inflammatory cells have defensemechanisms against invading microorganisms and are known to exert theirantimicrobial actions by releasing reactive oxygen species (ROS),proteolytic enzymes and other toxic metabolites. While these ROS andother toxic compounds damage pathogens and host cells together,antioxidant defense system such as superoxide dismutase (SOD), catalase,glutathione, and glutathione peroxidase also affect the host cellsspecifically.

Standard Method Vs Stochastic Confinement

The standard procedure for a patient presenting a bacterial infection inthe blood, is to take a blood sample from the patient, the blood samplehaving a cell density of 100 CFU/mL. If the patient has an infection ofmethicillin resistant Staphylococcus aureus, and traditional culturebased methods are used, detection of the Staphylococcus aureus takesabout 12 hours. At that point the susceptibility of the pathogen toantibiotics would not yet be known; antibiotic testing would requireanother 6 hours.

If the blood sample is stochastically confined into 0.1 mL plugs,detection takes less than 4 hours. Because stochastic confinement hasthe ability to screen individual bacterium, up to 100 differentantibiotic conditions may be screened from a 1 mL blood sample without apreincubation step. Detection time for bacteria with S. aureus decreasesby 1.5 hours for every order of magnitude increase in cell density attime zero (i.e. 100 CFU/mL takes 12 hours, 1000 CFU/mL take 9.5 hours).Therefore with confinement in nL plugs, concentration can be increasedfrom 100 CFU/mL to 10⁷ CFU/mL which decreases detection time by 7.5hours. (P. Kaltsas, S. Want and J. Cohen, Clin. Microbiol. Infect.,2005, 11, 109-114.) In 23-30% of all cases, an inappropriate antibioticis initially administered (S. D. Carrigan, G. Scott and M. Tabrizian,Clin. Chem., 2004, 50, 1301-1314.)

In the standard method, the infection is identified after 12 hours andthen it takes 6 additional hours to screen antibiotics. Many patientswould not receive appropriate antibiotic treatment for over 18 hours,which means that the infection has worsened and the chance of mortalityis greatly increased.

In the stochastic confinement method, infection and antibioticsensitivity of infection is known after 4 hours or less. Antibiotictreatment can begin much sooner. Other advantages of stochasticconfinement are the ability to run multiple tests on the sample withoutpreincubation, therefore the clinician can have an in depthcharacterization of the pathogen (antibiotic sensitivity, serotype,strain, genetic information, other functional tests like propensity forvirulence) within a few hours. Since confinement involves testingindividual cells, heterogeneity in the activity/phenotype within thepopulation of cells may be tested. For instance, it may be detected that1% of bacteria in the sample are resistant to oxacillin even though 99%of the cells are sensitive. A traditional method might not detect thisdifference. Consider starting with a sample at 100 CFU/mL in which only1% of cells are resistant. Detection time of resistant cells would befor a density of 1 CFU/mL. In the standard method, protocols for runningthe test might not be long enough (an additional 3 hours or more) toeven detect this type of resistance in the standard method. In addition,subpopulations of resistant cells typically grow more slowly, increasingthe chances of not detecting the cells in traditional tests.

Stochastic confinement is useful for large scale monitoring of resistantstrains in the population and offers many benefits. Bacterial infectionsin the hospital setting do not routinely undergo in depthcharacterization of the infecting strain. Stochastic confinementprovides a cheap and efficient method of characterizing pathogens sothat hospital bacterial infections can be characterized routinely in thehope of better controlling them. In addition, functional tests are abetter way to characterize pathogens than genetic tests because thegenetic marker for a new resistance mechanism can be found immediately.Therefore there is less of a time delay between identification a newresistance mechanism and the discovery of a genetic marker useful forthe diagnosis of the resistant strain. Stochastic confinement alsoenables increased tracking of resistance patterns leading to earlyrecognition of resistant strains and enables health care agencies totrack the spread of resistant strains.

Functional detection is possible as well wherein target or host cellsand potentially infectious organisms are added into plugs and monitoredfor infection. Human/mammalian, bird, live stock, plants/crops can allbe used as the target or host cells for these kinds of infectivityassays. The same techniques can be used to look for ways of reducinginfectivity of microbes in any of these systems. The term virulence isused to mean direct infectivity, or killing remotely, any kind ofpathogenicity, or negatively affecting the host cell in other ways orsporulating of bacterial spores, or activation of viral particles, ortransition of bacterial cells from dormant to active form (relevant totuberculosis). “Particle” refers to a cell, a viral particle, a spore,and the like.

The same ideas can be applied to general screening of cells and theiractivity such as switching from one state to another which may depend onthe concentration of soluble or surface-bound factors, or presence ofother cells. Examples include stem-cell differentiation and cancer cellactivation.

The hybrid approach is described in Liang Li, Debarshi Mustafi, QiangFu, Valentina Tereshko, Delai L. Chen, Joshua D. Tice, and Rustem F.Ismagilov, “Nanoliter microfluidic hybrid method for simultaneousscreening and optimization validated with crystallization of membraneproteins”, PNAS 2006 103: 19243-19248 as well as in publicationsincorporated by reference above.

An example of chemical systems capable of amplification is described inKastrup et al. PNAS 2006 Oct. 24; 103(43):15747-52 as well as inpublications incorporated by reference above. This simple chemical modelsystem, built by using a modular approach, can be used to predict thespatiotemporal dynamics of complex chemical networks. Microfluidics isused to create in vitro environments that expose both the complexnetwork and the model system to surfaces patterned with patchespresenting stimuli. Such chemical model systems, implemented withmicrofluidics, may be used to predict spatiotemporal dynamics of complexbiochemical networks.

Reducing virulence is a good strategy to fight microbial infections.Targeting virulence factors for screening antibacterial drugs is apotential way to develop novel drugs. To escape cross-resistance ofcurrent drugs, targeting to virulence factors involved in humanpathogenesis is considered essential. To find compounds that reducevirulence, conditions need to be presented that induce virulence so thatthe virulence and the effects of drugs on virulence can be monitored.There are a number of ways that pathogens cause disease in humansincluding adhesion, colonization, invasion, immune response inhibition,and toxins. Since pathogenic bacteria have different ways of inducingvirulence, conditions of inducing, monitoring, and evaluating virulenceshould be selected as the target.

Conditions that lead to virulence are often not known. Components mayinclude the presence of host's cellular signals, accumulation ofsecreted microbial factors, presence of surface-bound cellularcomponents and signals, presence of host cells, presence of molecularspecies associated the host's environment, presence of molecular speciespresent on surfaces of host cells, reactive oxygen species, or reactivenitrogen species.

One representative example of inducing virulence is the generation ofreactive oxygen species (ROS). Inflammatory cells show defense mechanismagainst invading microorganisms and are known to exert theirantimicrobial actions by releasing reactive oxygen species (ROS),proteolytic enzymes and other toxic metabolites. While these ROS andother toxic compounds damage pathogens and host cells together,antioxidant defense system such as superoxide dismutase (SOD), catalase,glutathione, and glutathione peroxidase protect the host cells. Bacteriamay detect the presence of ROS to control their virulence.

The bacterial detection method may be used to determine conditions thatinduce virulence using the hybrid method. Any of the factors that inducevirulence may be introduced into plugs containing microbial cells andtheir concentration varied. In addition, surface chemistries may be usedto incorporate molecules present on host's cell surfaces. These surfacechemistries may also be screened in the context of the hybrid and/orcartridge method. A combination of several cartridges, each containing adifferent set of reagents (e.g. solution-based and surface-based) may beused to screen combinations of reagents at different concentrations.Activation of virulence may be observed as a function of solution and/orsurface conditions, leading to determination of optimal conditionsand/or conditions that are most physiologically relevant. These methodsmay be used in combination with confinement.

An array of host cells may be introduced using the hybrid and/orcartridge method to determine the kinds or types of cells that amicroorganism may be virulent against. The hybrid and/or cartridgeapproach may be used to screen a wide range of microorganisms against aparticular host to determine which microorganisms may be virulentagainst the host.

Confinement, alone or in combination with other factors, may be used toinduce virulence. For example, confinement may lead to accumulation ofsecreted microbial factors turning on virulence. Virulence induced underconfinement may be more physiologically relevant.

Using the methods described herein, microbial cells or particlesisolated from a patient may be tested for their ability to inducevirulence. Hybrid and/or cartridge methods may be especially attractivefor such determination because they allow variation in the concentrationof the inducing factors.

Co-confinement of microbial cells and host cells (human/mammalian orbird/live stock/plants/crops hosts) may further improve induction ofvirulence, as both microbial and host factors may accumulate within theconfined volume. Such co-confinement may be created, for example, byeither forming plugs from at least two streams (one containing asuspension of microbes and the other a suspension of target cells), orby creating plugs containing one type of cells or particles (e.g.,microbial cells or viral particles), and injecting them with asuspension containing the other type of particle (e.g., target cells).

Changes in virulence may be monitored using a variety of methodsincluding assaying for the secretion of surface expression ofmolecules/proteins/enzymes associated with virulence including lipases,virulence factors, iron siderophores, and the like. The assay may betailored for the specific type of virulence mechanisms. For example, foradhesion, the ability of cells to stick to a host cell and detectingsecretion of molecules known to promote adhesion to other cells isimportant. In colonization and invasion, disruption of cell membranesand promotion of endocytosis is important and thus, injection systems toinject protein/genetic material into another cell (for example via thetype III secretion system) may be useful. For immune responseinhibitors, molecules that bind to antibodies, formation of capsulesaround the cell, induction of fibrin formation to surround the bacterialcell and prevention of recognition by the host are important. Fortoxins, molecules are released that may be detected using fluorescenceassays, agglutination assays, light producing reactions, color changereactions and the like. Also, production of certain factors associatedwith virulence (e.g. the lethal factor associated with B. anthracisvirulence) may be detected, or functional tests of the effects on thetarget cell may be used.

In terms of ROS, P. aeruginosa induction of oxidative stress in the hostcells may be assessed by measuring changes in lipid peroxidation andglutathione contents in the host cell line or tissues. In addition, theactivity of the antioxidant systems can be evaluated by measuringactivities of superoxide dismutase, catalase, and glutathioneperoxidase. (Microbial Pathogenesis, Volume 32, Issue 1, January 2002,pages 27-34).

If conditions leading to activation of virulence are known, or forexample once they are identified using methods described in thisapplication, a screen may be conducted for agents that reduce virulence.These agents may be small molecules, proteins, antibodies, etc. Thereare numerous applications to humans, livestock, plants, and the like.The method is especially useful for infections which remain dormant forlong periods of time and then cause problems periodically when virulenceis induced such as TB, malaria, and herpes. Some P. vivax and P. ovalesporozoites do not immediately develop into exoerythrocytic-phasemerozoites, but instead produce hypnozoites that remain dormant forperiods ranging from several months (6-12 months is typical) to as longas three years. After a period of dormancy, they became reactivate andproduce merozoites. Hypnozoites are responsible for long incubation andlate relapses in these two species of malaria.

Drugs may be compounds or entities that alter, inhibit, activate, orotherwise affect biological or chemical events. For example, drugs mayinclude, but are not limited to, anti-AIDS substances, anti-cancersubstances, antibiotics, immunosuppressants, anti-viral substances,enzyme inhibitors, including but not limited to protease and reversetranscriptase inhibitors, fusion inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, and imaging agents.

Possible applications include screening for drugs that reducevirulence/infectivity of a strain of bacteria and testing bacterialisolates from patients for virulence. For example, a hybrid screenand/or cartridge system pre-loaded with several drugs may be injectedinto plugs at the same time as combining infectable host cells withbacteria. Preventing organisms from virulence is less likely to causeevolution of resistance than simply killing them. Importantly,confinement may change virulence. Confinement may induce virulence notobserved in well-plates, so the method may have advantages overtraditional well-plate methods.

The sensitivity of a bacterial strain to many antibiotics can bescreened in a single experiment by using plug-based microfluidics. Asingle bacterial sample can be combined with many antibiotics togenerate an antibiogram, or chart of drug susceptibilty. A pre-formedarray of plugs of six antibiotics—two beta-lactams (ampicillin, AMP, andoxicillin, OXA); a cephalosporin (cefoxitin, CFX); a fluoroquinolone(levofloxicin, LVF); vancomycin, VCM; and a macrolide (erythromycin,ERT)—was generated by aspiration. Antibiotics were tested at thebreakpoint concentration, (British Society for AntimicrobialChemotherapy, BSAC Methods for Antimicrobial Susceptiblity Testing,2007) the accepted concentration of antibiotic at which bacterialsusceptibility is determined (FIG. 2 d). Since stochastic confinement ofthe bacterium into nanoliter-sized plugs generates many empty plugs, 50plugs of each antibiotic were generated such that it was statisticallylikely that each condition would contain several plugs each occupied bya single bacterium. In total, 400-500 plugs were formed for each screen,which consisted of 6 drug conditions and 2 blank conditions. All 400-500plugs were collected in the same coil of tubing. A blank condition waslocated at the beginning and end of the array to ensure that theposition in the array did not affect assay results. The plugs in thisantibiotic array were merged with Methicillin Resistant S. aureus (MRSA,ATCC#43300) at an initial cell density of 4×10⁵ CFU/mL and the viabilityindicator on-chip to form plugs approximately 4 mL in volume, asillustrated in FIG. 2 a. The merged plugs were collected and incubatedfor 7 h at 37° C. After incubation, the fluorescence intensity of theplugs was measured.

Occupied plugs containing an antibiotic to which the bacterial strainwas resistant showed increased fluorescence intensity, whereas plugscontaining an antibiotic to which the bacterial strain was sensitiveshowed no significant increase in fluorescence intensity (FIG. 2 c).Plugs containing VCM were used as a negative control, because VCMinhibited this S. aureus strain in macro-scale experiments, in agreementwith expectations (B. T. Tsuji, M. J. Rybak, C. M. Cheung, M. Amjad andG. W. Kaatz, Diagn. Microbiol. Infect. Dis., 2007, 58, 41-47). Theaverage increase in fluorescence from all plugs containing VCM was useda baseline to which the increase in fluorescence intensity of all otherplugs was compared (FIG. 2 b, Δ VCM). Four out of 49 (12%) control plugswith no antibiotic (FIG. 2 b, +blank 1) showed an increase influorescence intensity more than three times greater than the VCMbaseline, indicating that they were occupied by bacteria. However, theother plugs with no antibiotic showed an increase in fluorescenceintensity similar to the baseline, indicating that they were unoccupied(FIG. 2 b, +blank 1).

By comparing the fluorescence increase in each plug to the VCM baseline,it can determine which antibiotics were toxic to the bacteria. Plugsoccupied with a viable bacterium showed an increase in fluorescenceintensity greater than three times the VCM baseline. FIG. 2 c shows theaverage intensities of plugs that showed an increase in fluorescenceintensity greater than 3 times the baseline (black bars) and plugs thatshowed an increase in fluorescence intensity less than 3 times thebaseline (hatched bars). No plugs containing VCM or LVF had afluorescence increase greater than 3 times the baseline, indicating thatMRSA was sensitive to these antibiotics. Poisson statistics (Eq. 2) canbe used to predict the probability of not loading a bacterium into anyof plugs in the conditions LVF or VCM. In other words, Eq. 2 predictsthe possibility of the LVF or VCM results being false-negative.

$\begin{matrix}{{f\left( {k,\lambda} \right)} = \frac{\lambda^{k}^{- \lambda}}{k!}} & (2)\end{matrix}$

In Eq. 2, f is the probability of having k bacteria in a plug given anaverage bacterial loading of λ bacteria per plugs. The experimentallydetermined λ was 0.12, as 12% of control plugs with no antibioticsreceived bacteria (FIG. 2 b, +blank plugs). For k=0 and λ=0.12, wecalculated the probability of having an unoccupied plug to be 0.887. Theprobability of having 49 unoccupied plugs is 0.887⁴⁹, or 0.0028. Giventhat LVF and VCM had at least 49 plugs, the probability of afalse-negative due to loading is less than 0.3%.

The results from the MRSA antibiotic screen were used to make theantibiogram in FIG. 2 d. The antibiotics were tested at the breakpointconcentration, and the fluorescence data was used to determine if thebacterial strain was sensitive (S) or resistant (R) to the antibiotic.Sensitive means that no plugs containing a specific antibiotic showed anincrease in fluorescence intensity greater than 3 times the VCMbaseline. Resistant means that at least one plug containing a specificantibiotic showed increased fluorescence intensity greater than 3 timesthe VCM baseline. The susceptibility profile generated for MRSA by usingthe microfluidic screen was identical to the profile generated by usingMueller-Hinton agar plate tests and similar to previous reports in theliterature for MRSA. (B. T. Tsuji, M. J. Rybak, C. M. Cheung, M. Amjadand G. W. Kaatz, Diagn. Microbiol. Infect. Dis., 2007, 58, 41-47)However, antibiotic sensitivity testing is influenced by many factors,including bacterial load, culturing conditions, temperature, bacterialstrain, and type of assay used to detect sensitivity. In addition, acell population may contain sub-populations of cells with variablesensitivity to a given antibiotic. All of these factors should beconsidered and further characterized before formulating guidelines forimplementing plug-based antibiotic sensitivity assays.

Plug-based methods can also be used to determine the minimal inhibitoryconcentration (MIC) of an antibiotic against a bacterial sample.

Next, this microfluidic approach was used to determine the MIC of theantibiotic cefoxitin (CFX) for MRSA and MSSA (FIG. 6). This assay wassimilar to the antibiotic screening assay described above, except thatthe pre-formed array of antibiotic plugs all contained the sameantibiotic and the concentration of that antibiotic in each plug of thepre-formed array was different. Again, plugs containing saline solutionwere included at the beginning and end of the array to serve as negativecontrols and to ensure that the first and last plugs of the array gavesimilar assay results. The positive control plugs consisted of CFX at aconcentration of 24 mg/L, as both strains are known to be inhibited byCFX at this concentration. Plugs of the antibiotic array were mergedwith bacteria and the fluorescent viability indicator as illustrated inFIG. 6 a and incubated at 32° C. Plugs with MRSA were incubated for 6.75h and plugs with MSSA were incubated for 6.5 h. It should be noted thattemperature can affect the results of antibiotic sensitivity assays.Here, the difference in MIC of MRSA and MSSA was discerned by assaysconducted at 32° C.

After incubation, the fluorescence intensity of the plugs was measured.Here, the average increase in fluorescence intensity of plugs containing24 mg/L CFX was used as the baseline to which the increase influorescence intensity of other plugs was compared. Because MRSA isresistant to many beta-lactam antibiotics, CFX should be less effectiveagainst the strain MRSA. As expected, the MIC of CFX was higher for MRSA(<8 mg/L) than the MIC of CFX for MSSA (<4.0 mg/L) (FIG. 6 b). Theseresults validate the use of this plug-based technology for screeningboth the susceptibility and the minimal inhibitory concentration of manyantibiotics against a single bacterial sample.

The issue of screening media conditions along with drugs is of interest.The outcome of a drug screen can be heavily influenced by the media andculturing conditions used for the screen. To eliminate false negativesin a drug screen, it would be useful to screen the same drug conditionin a variety of media and culturing conditions. For instance, an unknownsample of bacteria may be screened against the drug oxicillin at 30 and37° C., 100 and 150 mM NaCl, and in Luria Bertani media and soytrypticase media. The media may: 1) influence the growth rate ofbacteria regardless of drug condition (if the media condition is suchthat bacteria grow very slowly, the assay would falsely determine thatthe drug is killing the bacteria); or 2) influence the interactionbetween the bacteria and the drug.

Stochastic confinement combined with plug-based microfluidic handlingmethods accelerates bacterial detection and enables rapid functionalantibiotic screening. By using this method, assays may be performed on asingle bacterium, potentially eliminating the need for pre-incubation.By confining and analyzing single bacterium in plugs, detection time isnow determined by plug volume. We were able to achieve detailedfunctional characterization of a bacterial sample in less than 7 hours.We also demonstrated that a bacterium in a 1 mL plug may be detected inas little as 2 hours. The detection time is limited by the formation andmeasurement of plugs of small volume and is less dependent on theinitial concentration and growth rate of bacteria in the sample. Thisfeature may be potentially important for accelerated detection ofslowly-growing species such as M. tuberculosis, a pathogen ofsignificant importance world-wide. (E. Keeler, M. D. Perkins, P. Small,C. Hanson, S. Reed, J. Cunningham, J. E. Aledort, L. Hillborne, M. E.Rafael, F. Girosi and C. Dye, Nature, 2006, 444 Suppl 1, 49-57) Here, wehave demonstrated a screen with 400-500 mL plugs. High-throughputscreens with more conditions and increased concentration of the samplewould require methods that can handle larger numbers of smaller plugs,including methods for automated sorting and analysis. (Y. C. Tan, Y. L.Ho and A. P. Lee, Microfluid. Nanofluid., 2008, 4, 343-348; D. Huh, J.H. Bahng, Y. B. Ling, H. H. Wei, O. D. Kripfgans, J. B. Fowlkes, J. B.Grotberg and S. Takayama, Anal. Chem., 2007, 79, 1369-1376; K. Ahn, C.Kerbage, T. P. Hunt, R. M. Westervelt, D. R. Link and D. A. Weitz, Appl.Phys. Lett., 2006, 88, 024104; M. Chabert and J.-L. Viovy, Proceedingsof the National Academy of Sciences, 2008, 105, 3191-3196) Uponincorporating such methods for handling and sorting large numbers ofplugs of small volume, this technique may be used for the detection ofbacteria in a sample at a cell density much lower than 10⁵ CFU/mL. Sincethe activity of single cells is being measured, it is conceivable thatdetecting the presence of even a single bacterium in a sample may befeasible.

Given that a typical 5 mL blood sample from a patient with bacteremiacontains a cell density of 100 CFU/mL, (L. G. Reimer, M. L. Wilson andM. P. Weinstein, Clin. Microbiol. Rev., 1997, 10, 444-7) this method iscapable of performing dozens of functional tests on such a sample.Patient-specific characterization of bacterial species would not onlylead to more rapid and effective treatment, but such an advance wouldalso enable in-depth characterization of bacterial infections at thepopulation level. Such detailed characterization may aid in tracking andidentifying new resistance patterns in bacterial pathogens. (S. K.Fridkin, J. R. Edwards, F. C. Tenover, R. P. Gaynes and J. E. McGowan,Clin. Infect. Dis., 2001, 33, 324-329; R. T. Horvat, N. E. Klutman, M.K. Lacy, D. Grauer and M. Wilson, J. Clin. Microbiol., 2003, 41,4611-4616) The principles of these methods, stochastic single-cellconfinement and multiple functional assays without samplepre-incubation, may also be applied to other areas, including performingfunctional tests on field samples, detecting contamination of food orwater, separating and testing samples with mixtures of species,measuring functional heterogeneity in bacterial populations, andmonitoring industrial bioprocesses.

Other Applications

Confinement affects may be of particular use in the detection of slowgrowing bacterial strains such as Mycobacterium tuberculosis (the strainwhich causes tuberculosis, TB). To grow a culture of M. tuberculosisusing traditional methods takes several weeks. Current tests for TB arebased on an immune response, but cannot distinguish between someone withan active infection and someone who has previous immunization. Othercurrent tests involve PCR based methods. Stochastic confinement shouldincrease the sensitivity of detection based assays and also remove theneed for a lengthy preincubation step before running the detection assaysince detection may be done with a single confined cell.

Confinement effects may be used to screen natural sources for candidateorganisms or their genes that perform functions of interest or generatemolecules of interest. Functions of interest include nitrogen fixation,carbon recycling, hydrocarbon production, pollutant degradation, solarenergy conversion, forming a symbiotic relationship with other microbes,producing a toxin that kills other organisms, produces light, producesan odor, generates electricity. Molecules of interest include drugcandidates, small molecule inhibitors, enzymes which degrade cellulose,enzymes which degrade pollutants, adhesives, electron transportmolecules, metal chelators, selective inhibitors of small molecules,catalysts, plasticizing agents, and proteases.

Some of these functions may not occur in large volume (low density ofcell type which performs function) samples or samples with mixtures ofmicrobes. One advantage of confinement is that individual microbes willbe able to function under high density conditions. This would be usefulfor rare cell types in a sample, as high density functions would not beoccurring in the original sample due to the rare cells being at a lowcell density. Such functions would not be occurring in a macroscalesample. Confinement may also enable rare cell types of initiate growthor increase the growth rate due to the high concentration of rare cellsin a small volume plug after confinement. This type of approach can beextended to rare cell types from mixed species samples by confining rarecell at high densities to enable more rapid growth of the rare cells dueto both increased inoculation density and eliminating competition withother strains in the original mixed sample. The effect of confinement insmall volumes is increased if the molecules accumulating in the plug areimmiscible in the carrier fluid (the fluid surrounding the plug). Usingimmiscible fluids around the plug will prevent released microbialproducts from diffusing out of the plug and hence the released productswill more quickly accumulate and achieve a higher concentration in theplug.

Other applications include detecting bacteria for applications inhomeland security and safety of the food chain and water. It is alsopossible to apply these methods of detection to the areas of sepsis,bioenergy, proteins, enzyme engineering, blood clotting, biodefense,food safety, safety of water supply, and environmental remediation.

The following patents and patent applications are hereby entirelyincorporated by reference in their entirety: WO 05-010169A2, U.S. Pat.No. 6,500,617, WO 2007-009082 A1.

A process of collecting a useful product from stochastically confinedcells may comprise: confining organisms, cells or particles (bythemselves, or with their enemies such as other bacterial or othercells), or with addition of stimulating chemicals; accumulating theirproducts (antibiotics or other potentially valuable enzymes); usingthese products for detection; using these products for further screening(for example, dilute and different concentrations and merging withsuspensions of other bacteria to see if those bacteria get killed off,or drip those dilutions into standard growth assay plates); using theaccumulated enzyme and assaying for function (as described in thisapplication, including but not limited to cellulose degradation,catalysts for synthesis, disruption of biofilms); and adding otherassays. When plugs are used for stochastic confinement, allowing anorganism to multiply inside a plug and then splitting the plug intodaughter plugs (where at least two daughter plugs contain daughterorganisms) provides an opportunity to perform multiple assays on clonesof the organism (including assays that cannot be performed on a singleorganism or in a single volume).

All of the methods and applications described herein may be done undercontrolled atmosphere using plugs/droplets, where fluorocarbon canenable transport of gases to the plug, and massively parallelsmall-scale incubations under controlled atmosphere can be performed.This may be useful for control of virulence, for hydrogen generation andfor using organisms that require controlled atmosphere (anaerobes,organisms that consume or produce methane or other hydrocarbons or H₂S,etc).

The methods described herein may also be used to detect fungi, archaeaand other organisms in a sample.

Test Strips

General Components of Test-Strips

A test strip comprises an amplification layer and may comprise one ormore of the following layers: a filtration wetting layer withselectivity, a detection layer with threshold and a layer of substratefor signal output. A signal produced by target bacteria, for example anenzyme, will turn on the amplification reactions in the amplificationlayer. Multiple amplification layers may be applied to achieve a highmagnitude of amplification. A substrate is used to detect the generationof control molecule or the output of the amplification in the system.

The amplification region may be regulated by a threshold mechanism. Themethods for amplification may be chosen from those described in thesection entitled “Amplification.” A detailed definition of threshold maybe found below.

The techniques described in this section aim to detect a small number ofmolecules or particles in a short time with a high resistance to noiseand background signals. To achieve such goals, each of these techniquesconsists of multiple modules. The two most important modules are theamplification process with positive feedback that produces a largeamount of substances in short time and the inhibitory mechanism. Theinterplay between these two processes sets up a threshold or athreshold-like behavior. In an ideal system, a threshold is theconcentration below which an input gives a background output and abovewhich an input gives a the signal output, where the two outputs areeasily distinguishable. To achieve most useful amplification, the signaloutput must be significantly (often by two orders of magnitude or more)different than the background output. The transfer function, thefunction of output versus input, may be a shifted ideal step function.However, in many cases, it is impossible to achieve an ideal threshold,but possible to achieve a threshold-like behavior, with which thetransfer function is similar to a step function but has a finite slopeat the transition region. A sigmoidal function or a similar function maybe used to describe such threshold-like behaviors. Another way to lookat threshold is the time to reach maximum possible output as a functionof input. With an ideal threshold, this function is infinite when theinput is below the threshold and reaches a constant small positive valuewhen the input is above the threshold. With a threshold-like behavior,this function is very large when the input is below the threshold. Asthe amount of input increases from the threshold, this functiondecreases rapidly and reaches a very small value (such as 10% or less ofthat at the threshold). In the most ideal case, at threshold, a changein the number of input molecules of 1 unit leads to a drastic change inthe output.

As long as the threshold is tuned properly, the amplification processmay selectively respond to only the active, target particles (molecules)even in the presence of a large excess of interfering particles(molecules). One or multiple amplification layers with threshold may beadded to increase the degree of amplification. Threshold response may beincorporated in the detection layer to limit false positives and falsenegatives. Diffusion of signal molecules and control molecules may berestricted on each layer by choosing the appropriate material. Thus,stochastic confinement may be applicable to test strips. For example, abacterium on the strip is confined by limited diffusion (the reagentsand products are not mixed on purpose), or the test strip may bestructured to restrict diffusion, for example when based on alumina ortrack-etched membranes.

FIG. 8 is a schematic description of a test strip with an amplificationsystem. Bacteria are brought into contact with a filtration wettinglayer. Enzymes (E) produced by target bacteria, for example in the scaleof pM or even lower, enter a detection layer with threshold and turn onthe reactions to generate control molecules (C). After proceeding toamplification layer, the signal will be amplified and the concentrationof control molecule increased to μM or mM. The control molecules willreact with chromogenic, fluorogenic or other substrate in the substratelayer to give a strong output signal. Output signal can also begenerated in any other ways.

A timer region may be added to the test strip as shown in FIG. 9. Thistimer region is an analogous reaction which is not detecting theanalyte, but instead demonstrating the reaction is running correctlyunder the current conditions (age of strip, temperature, pressure,humidity, presence of certain impurities, etc.). It also gives the userthe assurance that the strip is working and that they have waited longenough for the results. There may be one or multiple timer regions toindicate different parameters (age of strip, temperature, pressure,humidity, presence of certain impurities, etc.)

The timer region may have the same amplification system with reactionsof the same sensitivity as the detection region. It may be used to testfor false positive. However, the timer regions may utilize othertechniques for specific purposes. Some amplification systems may bepreviously loaded with a known amount of analyte which may be activatedupon the beginning of the test (e.g. by wetting).

Amplification

Amplification schemes using enzyme cascades are known. In particular,enzyme cascades may be modified to detect the type of molecule notnaturally associated with the enzymes. For example, the crab bloodcascade may be used to detect air pollutants by modifying enzymes at thebeginning of the cascade to detect a new type of input.

Cascade assay formats may be used. These detect an analyte through aprocess wherein, a first signal generating compound (i.e., SGC #1)produces a product that may be utilized by a second SGC #2 to produce aproduct which, e.g., may be utilized by a third SGC #3. The subjectcascade of products from SGC #1-3 results in amplification which resultsin a greater overall signal than may be achieved by any single SGC.

Many substances could be detected by a scheme which uses the same(generic) amplification mechanism. For different analyte, a differentstarter reaction is designed, but all starter reactions produce the sameoutput. For example, a detection scheme with the first step which isdesigned to detect a specific activity and the second steps which isable to amplify the product of the first step. An example is using thecoagulation cascade as the generic amplification mechanism, and a set ofstarted reactions that all produce an activator of thrombin. One wouldonly have to redesign the starter reaction to detect a new analyte

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that take place in the same plug. Thereare two general ways in which this may be performed. In the firstmethod, the product of the first reaction is reacted with, or bound by,a molecule which does not react with the substrate of the firstreaction. In a second, the coupled reaction will only proceed in thepresence of the product of the first reaction. For example, a geneticelement encoding a gene product with a desired activity may then bepurified by using the properties of the product of the second reactionto induce a change in the detectable properties of the genetic element.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalyzed reaction. The enzymeto catalyze the second reaction may either be translated in situ in theplug or incorporated in the reaction mixture prior to incorporation intoa plug. Only when the first reaction proceeds will the coupled enzymegenerate a product which may be used to induce a change in thedetectable properties of the genetic element. Stochastic confinementcould be used to increase local concentration and signal to noise ratioand to give advantages described elsewhere herein. The product of thefirst reaction could be a substrate, enzyme, or cofactor of the secondreaction, or promote the release of inhibition of the second reaction.More reaction steps could be used.

The concept of coupling may be elaborated to incorporate multipleenzymes, each using as a substrate which is the product of the previousreaction. This allows for selection of enzymes that will not react withan immobilized substrate. It may also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product. Furthermore an enzyme cascade systemmay be based for the production of an activator for an enzyme or thedestruction of an enzyme inhibitor. Coupling also has the advantage thata common selection system may be used for a whole group of enzymes whichgenerate the same product and allows for the selection of complicatedchemical transformations that cannot be performed in a single step.

Previously developed methods of detection include those usingnucleotide-based amplification (such as immuno-polymerase chain reaction(iPCR), (Adler, M.; Wacker, R.; Niemeyer, C. M., Sensitivity bycombination: immuno-PCR and related technologies. Analyst 2008, 133,(6), 702-718.) amplification based on allosteric catalysis, (Zhu, L.;Anslyn, E. V., Signal amplification by allosteric catalysis. AngewandteChemie-International Edition 2006, 45, (8), 1190-1196.) biobarcode,(Nam, J. M.; Stoeva, S. I.; Mirkin, C. A., Bio-bar-code-based DNAdetection with PCR-like sensitivity. Journal of the American ChemicalSociety 2004, 126, (19), 5932-5933; Nam, J. M.; Thaxton, C. S.; Mirkin,C. A., Nanoparticle-based bio-bar codes for the ultrasensitive detectionof proteins. Science 2003, 301, (5641), 1884-1886.) molecular beacon,(Li, J. W. J.; Chu, Y. Z.; Lee, B. Y. H.; Xie, X. L. S., Enzymaticsignal amplification of molecular beacons for sensitive DNA detection.Nucleic Acids Research 2008, 36, (6).) liposome-based amplification,(Edwards, K. A.; Baeumner, A. J., Liposomes in analyses. Talanta 2006,68, (5), 1421-1431.) and nanowire sensor. (Zheng, G. F.; Patolsky, F.;Cui, Y.; Wang, W. U.; Lieber, C. M., Multiplexed electrical detection ofcancer markers with nanowire sensor arrays. Nature Biotechnology 2005,23, (10), 1294-1301; Cui, Y.; Wei, Q. Q.; Park, H. K.; Lieber, C. M.,Nanowire nanosensors for highly sensitive and selective detection ofbiological and chemical species. Science 2001, 293, (5533), 1289-1292.)Each of these techniques has one or multiple disadvantages: slowresponse, vulnerability to noise and environmental degradation, lowdetection limit, and requirement for highly sophisticated and expensivefabrication.

The techniques described in this section aim to detect a small number ofmolecules or particles in a short time with a high resistance to noiseand background signals. To achieve such goals, each of these techniquesconsists of multiple modules. The two most important modules are theamplification process with positive feedback that produces a largeamount of substances in short time and the inhibitory mechanism. Theinterplay between these two processes sets up a threshold or athreshold-like behavior. In an ideal system, a threshold is theconcentration below which an input gives a background output and abovewhich an input gives a the signal output, where the two outputs areeasily distinguishable. To achieve most useful amplification, the signaloutput must be significantly (often by two orders of magnitude or more)different than the background output. The transfer function, thefunction of output versus input, may be a shifted ideal step function.However, in many cases, it is impossible to achieve an ideal threshold,but possible to achieve a threshold-like behavior, with which thetransfer function is similar to a step function but has a finite slopeat the transition region. A sigmoidal function or a similar function maybe used to describe such threshold-like behaviors. Another way to lookat threshold is the time to reach maximum possible output as a functionof input. With an ideal threshold, this function is infinite when theinput is below the threshold and reaches a constant small positive valuewhen the input is above the threshold. With a threshold-like behavior,this function is very large when the input is below the threshold. Asthe amount of input increases from the threshold, this functiondecreases rapidly and reaches a very small value (such as 10% or less ofthat at the threshold). In the most ideal case, at threshold, a changein the number of input molecules of 1 unit leads to a drastic change inthe output.

An elementary amplification method for detecting molecules includes: a)a set of molecules or materials that constitute an amplification processthat is capable of amplifying one or multiple components (termedoutput); b) a set of molecules or materials that provides an inhibitorymechanism to stop or slow down amplification by the amplificationprocess described in a) when the amount of the analyte is insufficient;c) an activating mechanism which, once sufficient analyte (termed input)is present, triggers the amplification process described in a); d) areadout process to provide an apparent signal (with visual signal as oneexample).

Inhibition in part (b) and the amplification process in part (a) set upa threshold or threshold-like behavior which gives very little or nooutput if the input is below the threshold and gives fast and abundantoutput if the input is above the threshold. This case is termed positivecontrast.

Alternatively, the system may be set up with the input providing amechanism to inhibit the amplification process. In such cases, with abelow-threshold amount of input, a lot of the output is produced, whilean above-threshold amount of input would give little or no output. Thisphenomenon is called negative contrast. The roles of part (b) and part(c) are swapped. The general feature is a big contrast (be it positiveor negative) between the output of a below- or above-threshold input.

Elementary amplification methods may be coupled in such a way thatoutputs of one may be used as inputs for another. This is also called acascade. The number of elementary amplification methods in a cascade maybe varied depending on how much more amplification is needed incomparison to what is provided by each elementary amplification method.

The analyte may be any molecules such as enzymes, DNA, RNA, smallmolecules, or any other molecules. It may come from any sourcesincluding bacterial components, human fluids, water samples, etc.

The amplification processes may be any reaction that is autocatalytic ora reaction network with one or more positive feedback loops. Theprocesses may involve enzymes such as those in the blood clottingcascade, apoptosis, and Limulus amebocyte (horseshoe crab) lysate (LAL),or any other enzymes. The processes may involve inorganic chemicals suchas the Co(III)-Co(II)-oxone system, (Endo, M.; Abe, S.; Deguchi, Y.;Yotsuyanagi, T., Kinetic determination of trace cobalt(II) by visualautocatalytic indication. Talanta 1998, 47, (2), 349-353; Endo, M.;Ishihara, M.; Yotsuyanagi, T., Autocatalytic decomposition of cobaltcomplexes as an indicator system for the determination of trace amountsof cobalt and effectors. Analyst 1996, 121, (4), 391-394; Tsukada, S.;Miki, H.; Lin, J. M.; Suzuki, T.; Yamada, M., Chemiluminescence fromfluorescent organic compounds induced by cobalt(II) catalyzeddecomposition of peroxomonosulfate. Analytica Chimica Acta 1998, 371,(2-3), 163-170.) Ag⁺-Ag(0) system, systems in which a metal surfacecatalyzes reduction of Ag+ or other ions, producing more metal surfaceavailable for catalysis the chlorite-iodide system, (Dateo, C. E.;Orban, M.; Dekepper, P.; Epstein, I. R., J. Am. Chem. Soc. 1982, 104, 2,504-509) or thiosulfate-chlorite-hydronium system. (Runyon, M. K.;Johnson-Kerner, B. L.; Ismagilov, R. F., Minimal functional model ofhemostasis in a biomimetic microfluidic system. AngewandteChemie-International Edition 2004, 43, (12), 1531-1536; Horvath, A. K.;Nagypal, I.; Epstein, I. R., Kinetics and mechanism of the chlorinedioxide-tetrathionate reaction. Journal of Physical Chemistry A 2003,107, (47), 10063-10068; Horvath, A. K.; Nagypal, I.; Epstein, I. R.,Oscillatory photochemical decomposition of tetrathionate ion. Journal ofthe American Chemical Society 2002, 124, (37), 10956-10957; Nagypal, I.;Epstein, I. R., Systematic Design of Chemical Oscillators 0.37.Fluctuations and Stirring Rate Effects in the Chlorite ThiosulfateReaction. Journal of Physical Chemistry 1986, 90, (23), 6285-6292.).They may also involve organic reactions, such as those with acid asautocatalysts. (Ichimura, K., Nonlinear organic reactions to proliferateacidic and basic molecules and their applications. Chemical Record 2002,2, (1), 46-55.) They may also be combinations of different types ofchemicals.

Phenomena in which nucleation is involved may also be used as anamplification process, in which a crystal of aggregate produced bynucleation may serve as a nucleus to promote more production of suchcrystal or aggregate.

An amplification process may also be achieved by using materials thatrelease substances that catalyze the release of more of such substances.

There are many inhibitory mechanisms. Each mechanism may occur directlyor indirectly through more than one reaction or processes.

The first mechanism may use chemical inhibitors. These chemicalinhibitors may be stoichiometric, such as those that bind to enzymes andblock the active sites or ligands that bind to and sequester metalcations. The chemical inhibitors may also be catalytic, such an enzymethat cleaves an active enzyme that is important in positive feedbackloops.

The second mechanism may use materials to mechanically separateimportant components of the amplification process. These materialsinclude, but are not limited to, vesicles containing liquid, particlesof solid or gel, and any combination of single-layer or multi-layerparticles or vesicles of one type or multiple types.

There are many activating mechanisms. For example, the input may be oneor multiple types of the substances that are inhibited chemically ormechanically by materials. The input may directly or indirectly producemore of the substances that are inhibited chemically or mechanically inthe inhibitory mechanism. The input may directly or indirectly interferewith the chemical or mechanical inhibition by competition with thesubstances being inhibited. The input may directly or indirectlyinterfere with the chemical or mechanical inhibition by chemicallymodifying the chemical inhibitors or the materials used to mechanicallyseparate the components of the amplification process.

The input generates a high local concentration of substances that mayhave functions described above. Stochastic confinement is an example ofthis category.

With such flexibility of activating mechanism, the methods may bedesigned to be applicable to many kinds of analytes with differentdesired degrees of specificity. For example, if an existing method needsmodifying to be applicable for detection of a different analyte, theactivating mechanism may be changed completely or may be adapted withsingle of multiple steps to use the analyte of interest to promote theproduction of the analyte of the existing method.

Readout Processes

One or more substances that are amplified or activated by theamplification process may promote the production of some form of easilydetectable readout. This readout may be a visual signal based on color.The color may come from any reaction that can generate color when theinput (over the threshold) is present. For example, an enzyme amplifiedin the amplification process cleaves a fluorogenic substrate or achromogenic substrate to give a fluorescent signal or color. Some otherexamples are pH indicators, reduction-oxidation potential indicators,and indicators for specific cations.

Alternatively, the readout may be a visual signal based on production ofaggregates (precipitates) or crystals from any method when the input(over the threshold) is present. These aggregates (precipitates) orcrystals may be the result of any process such as chemical reaction orproduction of paramagnetic substances that come together as solid in amagnetic field.

Examples of reactions that produce paramagnetic solids that may be usedin an amplification process include: 1) Ag⁺->Ag:Ag+ has electronconfiguration of d¹⁰, so it is diamagnetic. Ag has electronconfiguration of s¹d¹⁰, so it is paramagnetic. The autocatalyst is Ag(or its surface to be more detailed);

2) Guyard reaction: Mn(VII) (such as, KMnO₄) reacts with Mn(II) (somesoluble salt such as nitrate or chloride) and makes MnO₂ which is ablack paramagnetic powder. The autocatalyst is MnO₂ solid (its surface).(Polissar, M. J. Journal of Physical Chemistry 1935, 39, 1057.)

Further still, the readout may be any other detectable signal, such asan electrical signal that gives analog or digital readout or any otherkind.

Amplification Process Involving Enzymes with Chemical AmplificationProcesses

The amplification process may be an autocatalytic reaction or reactionnetwork that has a positive feedback. This positive feedback may beachieved through one step (FIG. 10). In such case, an autocatalyticenzyme may catalyze the cleavage of its precursor. (A chemical inhibitormay also used to inhibit this autocatalytic enzyme, and a fluorogenicsubstrate may be used to detect this autocatalytic enzyme). Positivefeedback may also be achieved through two steps (FIG. 11). In such case,enzyme 1 may catalyze the cleavage of enzyme precursor 2 to produceenzyme 2, which may catalyze the cleavage of enzyme precursor 1 toproduce enzyme 1. (Common or specific inhibitors and fluorogenicsubstrates for enzyme 1 and enzyme 2 may be included). Generally,positive feedback may be achieved through any number of steps, in whichone substance, called substance 1, may catalyze directly the productionof itself or indirectly by catalyzing the production of anothersubstance that may directly or indirectly catalyze the production of thesubstance 1. Many positive feedback loops may also be coupled with eachother into a cascade, in which the output of one positive feedback loopis used as the input of another (FIG. 12). In such case, the number ofpositive feedback loops with threshold must be at least 1 and may varyfrom 1 to all.

In certain embodiments at least one enzyme in the positive feedback loopis inhibited by an inhibitor. Not every enzyme is necessarily inhibited,but many may be. The inhibitors may be those found naturally orsynthesized. Antibodies may also be used as inhibitors.

If there is only one enzyme involved in the positive feedback loops(FIG. 10), the input may be that enzyme. If there are multiple enzymesinvolved in the positive feedback loops (FIG. 11), the input may besingle ones or combinations of the enzymes. The input may produce singleor combinations of enzymes from the same or different precursors as inthe amplification process, through one or many steps. The input may alsobe much more strongly binding substrate for the inhibitor, or promotesthe production of much more strongly binding substrate for theinhibitor. The input may chemically alter the inhibitors and disable theinhibitory effect, or promote the production of substances with suchfunction.

If fluorogenic substrates are used, one or multiple fluorogenicsubstrates may be cleaved by one or multiple enzymes in theamplification process, through one or multiple steps. In certainembodiments, there has to be at least one cleavage reaction but onecleavage reaction may or may not be sufficient. For methods with plasmaor whole blood, blood clotting may also be used as a visual readout.

Amplification Using Materials

Amplification process: The unit of the material contains molecules thatonce released can promote the release of more molecules from other unitsof the material. The units may be vesicles containing liquid (such asliposomes) or particles (of solid or gel).

Amplification using materials may be coupled with chemicalamplification. In other words, besides effects on the material, thereleased molecules may also undergo chemical amplification.

The molecules enclosed in the materials may be inactive and getactivated when released by chemical reactions with substances in thebulk or by any other ways such as change in conformation, release ofself-inhibition by an attached inhibitor, etc. They may also be in thesame form after release, but the breakage of the material is very slowwhen they are enclosed, such as when the material is designed to havedifferent reactivity with the molecules in the enclosed environment orin the bulk.

Inhibitory mechanism: These molecules may also be inhibited by somesubstances in the bulk when they are released.

Activating mechanism: The input may also be or produce (through one ormultiple steps) a substance that releases the molecules from thematerials (this substance may be similar to or different from theenclosed) or releases the molecules from the inhibitor (by competingwith the inhibitor or by inactivating the inhibitor).

Readout process: readout processes described elsewhere herein may beused, depending on specific situations.

Activating mechanism via generating a high concentration of substances.

In certain embodiments, the input generates a high local concentrationof substances.

This technique can be used in combination with amplification processesusing chemical reactions or materials.

Stochastic Confinement

Stochastic confinement as described above is a powerful tool to generatehigh local concentrations from a solution of low bulk concentration.This technique may also be used to distinguish interfering particles(molecules) with much weaker activity but much larger number than thoseof interest. When interfering particles (molecules, etc.) are in a largeexcess, the total activity of interfering particles may be higher thanthe total activity of active particles (molecules) of interest. Evenwith kinetic amplification via a threshold response, it is impossible todetect the signal from the particles of interest in the presence ofoverwhelming background signal from the interfering particles unlessseparation is used to isolate the particles of interest. This situationmay be commonly encountered in the microbiological analysis ofenvironmental samples. For example, human samples, including skin,saliva, and stool samples, being analyzed for microorganisms areroutinely contaminated with bacteria or other cells (blood). Stochasticconfinement is a powerful method to compartmentalize the activeparticles separately from the interfering particles. Therefore, as longas the threshold is tuned properly, the amplification process canselectively respond to only the active, target particles (molecules)even in the presence of a large excess of interfering particles(molecules).

Immobilizing Molecules onto a Limited Surface

Surfaces may be the interfaces of a plug, surface of a particle, surfaceof a microbe, and patterns on a surface which allows it to adsorbspecific molecules. Many techniques for immobilizing molecules onto asurface may be used. These techniques include but are not limited tousing antibodies, biotin/avidin interaction, andHis-tag/Ni²⁺/nitrolotriacetic acid (His-tag/Ni/NTA) interaction.

Using Particles Containing Molecules

In some embodiments, the molecules are enclosed in the particles. Whenneeded the particles release the molecules providing a burst of highconcentration of these molecules in solution. This process may or maynot be autocatalytic. In other words, the released molecules do not haveto promote the release of more molecules, although they may.

Stochastic confinement, immobilizing molecules onto a limited surfaceand using particles containing molecules, may be used in differentcombinations to generate even higher local concentrations. For example,if the analytes are bacteria (with or without background interferingbacteria), the specific antibodies for the bacteria of interest can beused to concentrate a reporter enzyme on the surface of the bacteria.The bacteria tagged with reporter enzymes are then stochasticallyconfined into plugs. These plugs are then merged with plugs containingvesicles containing secondary reporter enzymes. The reporter enzymestagged on the bacteria promotes the breakage of the vesicles, releasingan amplified number of secondary reporter enzymes. The reporter enzymesthen act as input for one of the amplification processes.

Using Membranes

To generate a high local concentration, a membrane can be used to keepone particular component on one side. For example, a membrane that isimpermeable to H⁺ but permeable to R⁻—H⁺ can be used. On one side of themembrane, an amplification process uses R⁻—H⁺ (which diffuses in fromthe other side of the membrane) as input to initiate the production ofH⁺ and amplifies the amount of H. Other substances and amplificationprocesses may be used with appropriate membranes as well.

Using Phase for Separation

Two sets of substances are separated into insoluble or immiscible phases(solid/solid, solid/liquid, or liquid/liquid). A shuttle substance maytransfer between two phases and speeds up the reaction in one or bothphases by fast reactions with positive feedback. For example, the systemcontains solid oxidant and iron particles in water. If the analyte isFe²⁺ (or Fe³⁺) or produces Fe²⁺ (or Fe³⁺), Fe²⁺ is oxidized by the solidoxidant to Fe³⁺, which oxidizes Fe(0) (from the iron particles) to Fe²⁺.Eventually, the solution contains a lot of Fe²⁺ (if the metal particlesare in excess) or Fe³⁺ (if the solid oxidant is in excess) which may bedetected by strongly colored indicators for Fe²⁺ or Fe³⁺.

EXPERIMENTAL Microfluidic Device Design and Fabrication

Microfluidic devices were fabricated by using soft lithography (Y. N.Xia and G. M. Whitesides, Annu. Rev. Mater. Sci., 1998, 28, 153-184.) asdescribed previously. (L. S. Roach, H. Song and R. F. Ismagilov, Anal.Chem., 2005, 77, 785-796; H. Song and R. F. Ismagilov, J. Am. Chem.Soc., 2003, 125, 14613-14619; L. Li, D. Mustafi, Q. Fu, V. Tereshko, D.L. L. Chen, J. D. Tice and R. F. Ismagilov, Proc. Natl. Acad. Sci.U.S.A., 2006, 103, 19243-19248.) Except where noted below, plugs werecollected in PFA or PTFE Teflon tubing (Zeus, Orangeburg, S.C.) with 150μm or 200 μm inner diameter (I.D.). The tubing was cut at a 45 degreeangle, inserted into the outlet of the microfluidic device up to theinlet junction, and sealed into the device by using PDMS prepolymer(10:1 elastomer to curing agent). To aid in imaging of the plugs, theTeflon tubing was wound in a spiral on a glass slide, and PDMSprepolymer was poured over the tubing to fix it in place. The devicewith attached tubing was then autoclaved at 135° C. for 10 min tosterilize. Once sterilized, the glass slide containing the tubing wastransferred to a sterile Petri dish.

Flowing Solutions into the Microfluidic Devices

All solutions were loaded into 1700 series Gastight syringes (Hamilton,Reno, Nev.) with removable 27 gauge needles and 30 gauge Teflon tubing(Weico, Wire & Cable, Edgewood, N.Y.). To maintain sterility, thesyringes were filled and attached to the device within a biosafetycabinet. Syringes were connected to the microfluidic devices by using 30gauge Teflon tubing. Solutions where flowed into the microfluidicdevices by using previously described methods. (L. Li, D. Mustafi, Q.Fu, V. Tereshko, D. L. L. Chen, J. D. Tice and R. F. Ismagilov, Proc.Natl. Acad. Sci. U.S.A., 2006, 103, 19243-19248) Flow rates werecontrolled by using PHD 2000 infusion syringe pumps (Harvard Apparatus,Holliston, Mass.).

Bacterial Cell Culture

Cells were obtained from ATCC (Staphylococcus aureus ATCC#25923 (MSSA)and Staphylococcus aureus ATCC#43300 (MRSA)). Stock solutions of thecells were made by using Luria-Bertani media Miller formulation (LB)(BD, Sparks, Md.) containing 30% (v/v) glycerol and stored at −80° C.For each experiment, a vial of frozen stock was brought to roomtemperature and streaked onto a Modified Trypticase Soy Agar (TSA II,BD, Sparks, Md.) plate and incubated overnight at 30° C. Colonies fromthe plates were transferred to LB and cultured at 37° C., 140 rpm for 3h at which point OD₆₀₀ was 1.5-2.0. Cell densities were then adjusted bydiluting in LB. To maintain sterility, all procedures were performed ina biosafety cabinet and all tubing, devices, syringes, and solutionsused were either autoclaved, sterilized by EtOH, packaged sterile, orfiltered through a 0.45 μm PES or PTFE filter.

Antibiotic Preparation

Antibiotic stock solutions of ampicillin (AMP), oxacillin (OXA),cefoxitin (CFX), levofloxacin (LVF), vancomycin (VCM), erythromycin(ERT) were made by using 150 mM NaCl_(aq) at a concentration of 4000times greater than the final concentration in the plugs, filtersterilized, and then frozen at −80° C. (AMP, Fisher Bioreagents, FairLawn. NJ; OXA, LVF, Fluka, Buchs, Switzerland; CFX, VCM, ERT, Sigma, St.Louis, Mo.). For example, AMP was tested at the breakpoint concentrationof 0.25 mg/L, meaning that the stock solution was prepared at aconcentration of 1000 mg/L. In the case of erythromycin (ERT), a stocksolution was prepared at 1000 times the final concentration in plugs.Before each experiment, vials of the antibiotics were thawed and diluted1000×(250× for ERT) with saline containing 80 μM fluoresceincarboxylate. Fluorescein carboxylate was used to aid in indexing theresultant array of plugs. The plugs in FIG. 7 contain no fluoresceincarboxylate, since indexing was not required. The blank conditionsconsisted of 150 mM NaCl. Antibiotic solutions were further diluted onchip 1:3 (v/v) during plug formation. 20 μM fluorescein carboxylate didnot interfere with the viability assay, the activity of the cells, oreffectiveness of antibiotic in tests performed on 96 well plates.

Antibiotic Testing on Plates

Plates were made from Mueller Hinton Agar (Fluka, Switzerland). Afterautoclaving, the agar was cooled and antibiotics were added and 20 mLplates were poured. For CFX and OXA testing, 50 μL of MRSA and MSSAbacterial culture at 4×10³ CFU/mL was spread onto separate TSA plates.The plates were incubated at 30° C. After 16.5 h and 40 h the plateswere examined for colonies. MRSA colonies appeared on CFX after 16.5 hand on the OXA plates after 40 h. Even after 40 h, MSSA colonies did notappear on the CFX or OXA plates. For AMP, ERT, LVF, and VCM, 5 μL ofculture at 2×10⁴ CFU/mL were spread onto plates, and the plates wereincubated at 37° C. for 12 h. After 12 h, growth of colonies on theplates was considered resistance to the antibiotic and no colonies onthe plates were considered sensitivity to the antibiotic. For all tests,control plates with no antibiotic were inoculated to ensure that eachplate tested received many CFU during inoculation.

Comparing Detection Times of Bacteria in Nanoliter Plugs andMilliliter-Scale Culture

Plugs were formed by using the general methods described previously. (L.Li, D. Mustafi, Q. Fu, V. Tereshko, D. L. L. Chen, J. D. Tice and R. F.Ismagilov, Proc. Natl. Acad. Sci. U.S.A., 2006, 103, 19243-19248; D. N.Adamson, D. Mustafi, J. X. J. Zhang, B. Zheng and R. F. Ismagilov, LabChip, 2006, 6, 1178-1186) Plugs were formed in a 3 inlet PDMS devicewith 100 μm wide channels by flowing S. aureus culture in LB at 2×10⁵CFU/mL at 1 μL/min, a 20% alamarBlue solution in saline at 1 μL/min, andfluorinated carrier fluid at 5 μL/min. 25 plugs were collected in thechannel. Inlets and the outlet were sealed with silicon grease and thedevice was placed in a Petri dish containing LB for incubation. The sameaqueous solutions were mixed 1:1 (total volume 0.6 mL) in a 14 mLpolypropylene round-bottom tube (BD Falcon, Franklin Lakes, N.J.). After2.8 h, plugs were made from the milliliter-scale culture by using thesame method, with the cell culture containing alamarBlue for bothaqueous inlets. Both sets of plugs were immediately imaged by using aepi-fluorescence microscope (IRE2, Leica) with a Cy3 (Chroma 41007, Cy3)filter and a 10×0.3 NA objective for a 5 ms exposure time with binningset to 4 and gain set to 200. Fluorescent images of plugs were processedby subtracting the average background intensity from all images. Linescans (FIG. 1 b) with a width of 25 pixels were taken along the longaxis of each plug.

Experiment to Compare Plug Size to Detection Time

PDMS devices with channel widths ranging from 200 to 800 μm wereprepared. Teflon tubing with diameter similar to that of the channel wascut at a 45° angle, inserted into the device up to the inlet junction,and sealed in place using PDMS. For FIGS. 1 c and d, plugs were formedas above, with the exception that the ˜1500 mL plugs were made viaaspiration by using a manual aspirator. In addition, 1 mL plugs wereformed in PTFE tubing with an outer diameter (OD) of 200 μm and an innerdiameter (ID) of 90 μm, 690 mL plugs were formed in PTFE tubing with anOD of 700 μm and an ID of 600 μm, 100 and 120 mL plugs were formed inPTFE tubing with an OD of 800 μm and an ID of 400 μm, and 1500 mL plugswere formed in PTFE tubing with an OD of 1100 μm and an ID of 1000 μm.Plugs were collected in the Teflon tubing, the tubing was sealed withwax, and the tubing was placed in a Petri dish containing LB forincubation and imaging. Incubation and imaging was performed in amicroscope incubator (Pecon GmbH, Erbach, Germany). Time zero is definedas the time at which the sample entered the incubator, which was lessthan 20 min after sample preparation. Fluorescence measurements weretaken with 5 ms exposure times with a 5×0.15 NA objective using a 1×camera coupler for plug sizes 1, 12.6, 100, and 690 mL plugs and a 0.63×camera coupler for 1500 mL plugs. Plugs 125 mL in volume were imagedwith 10 ms exposure times with a 5×0.15 NA and a 0.63× camera coupler.

Plugs were analyzed by first separating them from the background bythresholding to exclude intensity below 250. The average intensity ofthe thresholded plugs was measured. Over time, the intensity of theplugs diverged into 2 groups, occupied plugs and unoccupied plugs. Alloccupied plugs had a change in intensity more than 2 fold unoccupiedplugs. Detection time is defined as the time at which the fold change ofthe occupied plugs compared to the intensity of unoccupied plugs reachesa maximum. Fold change in intensity is defined as the change inintensity of an occupied plugs divided by the average change inintensity of unoccupied plugs (Eq. 1).

Fold change_((t=ti))=Occupied plug(I _(t=ti) −I _(t=1))/Unoccupiedplugs(I _(t=1) −I _(t=1))  (1)

In Eq. 1, I_(t=ti) is intensity at time point i. The intensity of theempty plugs is the average of all empty plugs in each experiment.

Comparing Detection Times of Bacteria in Nanoliter Plugs and 96 WellPlates

For FIG. 1 d, 96 well plates results for FIG. 1 d were acquired in aTecan Safire II plate reader (MTX Lab Systems, Vienna, Va.) with Ex/Em560/630 nm, gain 25, and 40 μs integration time. 200 μL of cell culturesuspended in LB with 10% alamarBlue was added to wells of a Costar 96well assay plate with black sides and a clear, flat bottom (Corning,Corning, N.Y.). Each data point represents triplicate measurements takenat 37° C. Fold change in intensity from 96 well plate results werecalculated by using Eq. 1 where the well with LB and alamarBlue only wasthe unoccupied plug condition.

Screening Susceptibility of Bacteria to Many Antibiotics

For antibiotic screening experiments (FIG. 2), an array of ˜50 nLantibiotic plugs was aspirated into Teflon tubing (200 μm ID) using amanual aspirator. Air spacers were included between each antibiotic plugto prevent merging of adjacent antibiotic plugs and to enable indexingof plugs in the output array. Plugs of saline solution were included asthe first and last plugs in the preformed array to serve as positivecontrols. The Teflon tubing containing the array of antibiotic plugs wassealed into a device inlet by using wax (Hampton Research, Aliso Viejo,Calif.). To screen the susceptibility of MRSA and MSSA to eachantibiotic, bacterial samples and indicator were merged with thepreformed array of antibiotic. Bacterial samples were at a density of4×10⁵ CFU/mL in LB, and viability indicator solution was made by mixing4 parts alamarBlue solution (AbD Serotec, Oxford, UK) with 6 parts 150mM NaCl., The flow rate of the antibiotic array was 0.25 μL/min; theflow rate of the bacterial solution was 0.5 μL/min, and the flow rate ofthe viability indicator was 0.25 μL/min. The carrier fluid was FC40(Acros Organics, Morris Plains, N.J.) with a flow rate of 1.6 μL/min.For each antibiotic plug in the preformed array, approximately 50smaller plugs (4 mL in volume) were formed, each potentially containinga single bacterium. The resulting plugs were collected in the coil PTFETeflon tubing (I.D.=150 μm).

After plug formation, the tubing was disconnected from the PDMS device,and the ends were sealed with wax. The Petri dish containing the tubingwas filled with 20 mL of LB solution to prevent evaporation of the plugsduring incubation. The plugs were immediately transferred to amicroscope incubator (Pecon GmbH, Erbach, Germany). Time zero is definedas the time which the plugs entered the incubator, which was about 20minutes after plugs were formed. Fluorescence measurements for plugswere recorded by using an inverted epi-fluorescence microscope (DMI6000,Leica, Bannockburn, Ill.) with a 10×0.3 NA objective (HCX PL Fluotar)coupled to a CCD camera ORCA ERG 1394 (12-bit, 1344×1024 resolution)(Hamamatsu Photonics) by using a 0.63× camera coupler. Images were takenof each plug using Metamorph Imaging Software (Molecular Devices,Sunnyvale, Calif.) every 30 min with exposure times of 5 ms. Plugs wereanalyzed by first separating them from the background by thresholding toexclude intensity below 250. The average intensity of the thresholdedplugs was measured. The change in intensity at time point ti isI_(t=ti)−I_(t=1). In the experiment described in FIG. 2 c, fluorescenceintensity of plugs was normalized by setting the intensity of thebrightest plug to 100.

Determining the Minimal Inhibitory Concentration of a Drug Against aBacterial Sample

For MIC determination in plugs (FIG. 3), a procedure similar toscreening susceptibility of many antibiotics was used. The input arrayof antibiotics consisted of plugs of CFX at a range of concentrations.Bacterial samples were MRSA or MSSA in LB at cell densities near 10⁶CFU/mL. In FIGS. 3 b and c, fluorescence intensity of plugs wasnormalized as described for FIG. 2 c.

Statistical Analysis of Antibiotic Screening Results

Unpaired t-tests were performed to compare antibiotic screening resultsto positive and negative controls. For FIG. 2 c: VCM and LVF arestatistically different than positive controls and AMP, CFX, OXA, ERT,and blank conditions were all statistically different than the negativecontrol. For FIG. 3 b: 8 and 24 mg/L CFX were statistically differentthan positive controls and 0, 0.2, 1, 2, and 4 mg/L were statisticallydifferent than the negative control. For FIG. 3 c: 4, 8, and 24 mg/L CFXwere statistically different than positive controls and 0, 0.2, 1, and 2mg/L were statistically different than the negative control. P valuesare two-tailed.

Detection and Drug Screening of MRSA and MSSA in Human Blood Plasma

For FIG. 4, cells were suspended in a 1:1 mixture of human blood plasma(Pooled normal plasma George King Bio-Medical, Overland Park, Kans.) andLB containing 40% alamarBlue. Plugs were formed and collected in Teflontubing (200 μm ID). Images were taken with a 5×0.15 NA objective with a0.63× camera coupler. Texas red pictures were taking every 10 minuteswith exposure times of 25 ms. A bright-field image was taken atbeginning and end of experiment. Linescans of original plug images weretaken at time 0 and time 7.5 h. Adobe Photoshop was used to enhancecontrast of plugs shown in FIG. 4.

Microfluidic bacterial detection and drug screening are applicable tocomplex, natural matrices, including human blood plasma.

To validate the applicability of this method to detecting bacteria innatural matrices, this method was used to detect bacteria in a sample ofhuman blood plasma. Bacterial strains MSSA or MRSA were inoculated intopooled human blood plasma at a concentration of 3×10⁵ CFU/mL. To testthe sensitivity of the bacteria to beta-lactams, the antibioticampicillin (AMP) was added to the culture at the breakpointconcentration. The inoculated plasma was then combined on-chip withviability indicator as illustrated FIG. 4 a. After 7.5 h of incubationat 37° C., plasma samples infected with MRSA were distinguishable fromsamples infected by MSSA by screening the samples against AMP at thebreakpoint concentration. While plugs containing MRSA and AMP showed asimilar increase in fluorescence intensity to plugs containing MRSA andno AMP (FIGS. 4 b and c), plugs containing MSSA and AMP showed noincrease in fluorescence intensity (FIGS. 7 d and e).

Amplification examples 1-6 involve blood coagulation proteins.Amplification example 7 involves proteins in apoptosis. Amplificationexample 8 involves Limulus amebocyte lysate from Horseshoe crabs.

Amplification Example 1

Amplification process: The enzyme precursor is engineered prothrombinthat may be cleaved by thrombin to produce more thrombin. The potentialof this engineering approach is supported by a method to make anengineered factor X that may be cleaved by thrombin. (Louvain-Quintard,V. B.; Bianchini, E. P.; Calmel-Tareau, C.; Tagzirt, M.; Le Bonniec, B.F., Thrombin-activable factor X re-establishes an intrinsicamplification in tenase-deficient plasmas. Journal of BiologicalChemistry 2005, 280, (50), 41352-41359.)

Inhibitory mechanism: The inhibitor is hirudin (or other inhibitors orantibodies of thrombin such as antithrombin III, heparin, or anophelin),which binds to thrombin and prevent the cleavage of prothrombin bythrombin.

Activating mechanism: The analyte, bacterial phosphatase, cleaves a tagattached to an inhibitor of hirudin (anti-hirudin), allowing thisactivated molecule to inhibit hirudin and release thrombin from hirudin.Alternatively, the bacterial phosphatase cleaves a tag attached tothrombin. In both cases, thrombin then cleaves prothrombin and producesmore thrombin. The specificity of this method may be designed to matchexpectation by changing the specificity of the cleavage of the tag bybacterial phosphatase. If detection of an enzyme different fromphosphatase is needed, a different tag is used.

Readout process: Thrombin enzymatically cleaves a fluorogenic substrate(such as Boc-Asp(OBzl)-Pro-Arg-MCA) to give fluorescent signal.

This method is predicted to be feasible with concentration ofprothrombin from 1 nM to 1 μM, be activated with concentration ofthrombin input after concentrating techniques of as low as 10 μM, giveresponse after 1-10 minutes, and give amplification gain of 3 to 9orders of magnitude.

Amplification Example 2

Amplification process: The enzyme precursor is factor XII that may becleaved by factor XIIa to produce more factor XIIa in the presence ofdextran sulfate or negatively charge surface in general. (Tankersley, D.L.; Finlayson, J. S., Kinetics of Activation and Autoactivation of HumanFactor-Xii. Biochemistry 1984, 23, (2), 273-279.)

Inhibitory mechanism: The inhibitor is ecotin (or other inhibitors orantibodies of factor XIIa).

Activating mechanism: the analyte, bacterial phosphatase, cleaves a tagattached to an inhibitor of ecotin, allowing this activated molecule toinhibit ecotin and release factor XIIa from ecotin. The system may alsobe designed so that the analyte cleaves a tag attached to factor XIIa orkallikrein. Factor XIIa or kallikrein then cleaves factor XII andproduces more factor XIIa. The specificity of this method may bedesigned to match expectation by changing the specificity of thecleavage of the tag by bacterial phosphatase. If detection of an enzymedifferent from phosphatase is needed, a different tag is used.

Readout process: Factor XIIa enzymatically cleaves a fluorogenicsubstrate (such as Boc-Gln-Gly-Arg-MCA) to give fluorescent signal.

This method is predicted to be feasible with concentration of factor XIIfrom 0.1 μM to 10 μM, be activated with concentration of kallikreininput after concentrating techniques of as low as 1 nM, give responseafter 1-10 minutes, and give amplification gain of 3 to 5 orders ofmagnitude.

Amplification Example 3

Amplification process: The enzyme precursor is factor XI that may becleaved by factor XIa to produce more factor XIa in the presence ofdextran sulfate or negatively charge surface in general. (Gailani, D.;Broze, G. J., Factor-Xi Activation in a Revised Model ofBlood-Coagulation. Science 1991, 253, (5022), 909-912; Naito, K.;Fujikawa, K., Activation of Human Blood-Coagulation Factor-XiIndependent of Factor-Xii-Factor-Xi Is Activated by Thrombin andFactor-Xia in the Presence of Negatively Charged Surfaces. Journal ofBiological Chemistry 1991, 266, (12), 7353-7358.)

Inhibitory mechanism: The inhibitor is aprotinin (or other inhibitors orantibodies of factor XIa).

Activating mechanism: the analyte, bacterial phosphatase, cleaves a tagattached to an inhibitor of aprotinin, allowing this activated moleculeto inhibit aprotinin and release factor XIa from aprotinin. The systemmay also be designed so that the analyte cleaves a tag attached tofactor XIa. Factor XIa then cleaves factor XI and produces more factorXIa. The specificity of this method may be designed to match expectationby changing the specificity of the cleavage of the tag by bacterialphosphatase. If detection of an enzyme different from phosphatase isneeded, a different tag is used.

Readout process: Factor XIa enzymatically cleaves a fluorogenicsubstrate (such as Boc-Glu(OBzl)-Ala-Arg-MCA) to give fluorescentsignal.

This method is predicted to be feasible with concentration of factor XIfrom 0.1 μM to 10 μM, be activated with concentration of factor XIainput after concentrating techniques of as low as 1 nM, give responseafter 1-10 minutes, and give amplification gain of 3 to 5 orders ofmagnitude.

Amplification Example 4

Amplification process: Factor XII and prekallikrein are precursors offactor XIIa and kallikrein, respectively. In the presence of dextransulfate or a negatively charged surface in general, factor XIIa cleavesboth factor XII and prekallikrein to produce factor XIIa and kallikrein,respectively, while kallikrein cleaves factor XII to produce factorXIIa. (Tankersley, D. L.; Finlayson, J. S., Kinetics of Activation andAutoactivation of Human Factor-Xii. Biochemistry 1984, 23, (2),273-279.)

Inhibitory mechanism: Inhibitors or antibodies that are common to bothfactor XIIa and kallikrein (such as ecotin) or different inhibitorsspecific to each may be used.

Activating mechanism: the analyte, bacterial phosphatase, cleaves a tagattached to an inhibitor of ecotin, allowing this activated molecule toinhibit ecotin and release factor XIIa and kallikrein from ecotin. Thesystem may also be designed so that the analyte cleaves a tag attachedto factor XIIa and/or kallikrein, which then activate the amplificationprocess. The specificity of this method may be designed to matchexpectation by changing the specificity of the cleavage of the tag bybacterial phosphatase. If detection of an enzyme different fromphosphatase is needed, a different tag is used.

Readout process: Factor XIIa or kallikrein or both enzymatically cleavefluorogenic substrates (such as Boc-Gln-Gly-Arg-MCA for factor XIIa andPro-Phe-Arg-MCA for kallikrein) to give fluorescent signal.

This method is predicted to be feasible with concentration of factor XIIfrom 0.1 μM to 10 μM and concentration of kallikrein from 10 μM to 10nM, be activated with concentration of kallikrein input afterconcentrating techniques of as low as 1 nM, give response after 1-10minutes, and give amplification gain of 3 to 5 orders of magnitude.

Amplification Example 5

Amplification process: The reaction network with positive feedback isshown in FIG. 13. This is an example of cases in which positive feedbackloops may be achieved through multiple steps and many positive feedbackloops may be coupled with each other to form a cascade. In this network,there are two positive feedback loops. The first one includes theproduction of factor Xa from factor X catalyzed by the input (InhA1),the production of factor VIIIa from factor VIII, the binding of factorVIIIa to factor IXa to form a complex, and the production of Xa fromfactor X catalyzed by the VIIIa:IXa complex. The output of this firstloop is factor Xa. The second positive feedback loop includes theproduction of factor Va from factor V catalyzed by the input (factorXa), the binding of factor Va to the input (factor Xa), the productionof thrombin from prothrombin catalyzed by the Xa:Va complex or the inputof the first loop (InhA1), and the production of factor Va from factor Vcatalyzed by thrombin. The output of this loop is thrombin. Thrombin isdetected by the cleavage of a fluorogenic substrate to releasefluorescent molecules catalyzed by thrombin. ATIII/heparin is used toinhibit factor Xa, the VIIIa:IXa complex, and thrombin.

Inhibitory mechanism: The chemical inhibitors are ATIII/heparin that mayinhibit factor Xa, the VIIIa:IXa complex, and thrombin. Other common orspecific inhibitors for these enzymes, of for factor VIIIa, Va, and IXamay be used as well.

Activation mechanism: Similar to examples 1-4, the analyte, bacterialphosphatase, may cleave tagged molecules and release them. Thesemolecules may be the activated factors shown in FIG. 13 or inhibitors ofthe inhibitors of those activated factors used in the inhibitorymechanism. The specificity of this method may be designed to matchexpectation by changing the specificity of the cleavage of the tag bybacterial phosphatase. If detection of an enzyme different fromphosphatase is needed, a different tag is used.

Readout process: Thrombin enzymatically cleaves fluorogenic substrates(such as Boc-Asp(OBzl)-Pro-Arg-MCA) to give fluorescent signal as shownin FIG. 13. Other single of combinations of activated factors may beused to cleave fluorogenic substrates as well.

Using the techniques described in this application, this method ispredicted to be feasible with concentration of enzyme precursors from0.1 μM to 1 μM, be activated with concentration of InhA1 input afterconcentrating techniques of as low as 1 nM, give response after 1-10minutes, and give amplification gain of 3 to 6 orders of magnitude.

Amplification Example 6

This example is similar to amplification example 5, but is morecomplicated. Here the amplification process contains most of thecomponents in the natural blood clotting network, as shown in a review.(Kastrup, C. J.; Runyon, M. K.; Lucchetta, E. M.; Price, J. M.;Ismagilov, R. F., Using chemistry and microfluidics to understand thespatial dynamics of complex biological networks. Accounts of ChemicalResearch 2008, 41, (4), 549-558.) Positive feedback loops are achievedthrough multiple steps. Inhibitory mechanism, activating mechanism, andreadout process are also similar to amplification example 5, and can bedone with single of combinations of enzymes. Additionally, bloodclotting may also be visualized by eyes. (Song, H.; Li, H. W.; Munson,M. S.; Van Ha, T. G.; Ismagilov, R. F., On-chip titration of ananticoagulant argatroban and determination of the clotting time withinwhole blood or plasma using a plug-based microfluidic system. AnalyticalChemistry 2006, 78, (14), 4839-4849.)

Using the techniques described in this application and the reactionconditions previously described, (Kastrup, C. J.; Runyon, M. K.; Shen,F.; Ismagilov, R. F., Modular chemical mechanism predicts spatiotemporaldynamics of initiation in the complex network of hemostasis. Proceedingsof the National Academy of Sciences of the United States of America2006, 103, (43), 15747-15752.) the predicted response is in 1-5 minutes.

Results from simulations and experiments (Kastrup, C. J.; Runyon, M. K.;Shen, F.; Ismagilov, R. F., Modular chemical mechanism predictsspatiotemporal dynamics of initiation in the complex network ofhemostasis. Proceedings of the National Academy of Sciences of theUnited States of America 2006, 103, (43), 15747-15752.) shown in FIGS.14 and 15 support the ideas in amplification examples 1-6 discussedabove. In general, they show a threshold-like behavior in which the timeof response (time for amount of some certain substance to reach adetectable value) drastically reduces as the amount of input increasesover a certain value. Although only amplification examples 1, 2, and 4were considered in FIG. 14, and amplification example 6 in FIG. 15, thereaction network of amplification example 3 is similar to that ofamplification example 1 and the reaction network of amplificationexample 5 has complexity between those of amplification examples 4 and6. Therefore, amplification examples 3 and 5 is expected to work aswell.

FIG. 14 is the time to get response versus amount of input obtained bysimulation using previously found rate constants (Tankersley, D. L.;Finlayson, J. S., Kinetics of Activation and Autoactivation of HumanFactor-Xii. Biochemistry 1984, 23, (2), 273-279; Kuharsky, A. L.;Fogelson, A. L., Surface-mediated control of blood coagulation: The roleof binding site densities and platelet deposition. Biophysical Journal2001, 80, (3), 1050-1074; Ulmer, J. S.; Lindquist, R. N.; Dennis, M. S.;Lazarus, R. A., Ecotin Is a Potent Inhibitor of the Contact SystemProteases Factor Xiia and Plasma Kallikrein. Febs Letters 1995, 365,(2-3), 159-163. Kawabata, S. I.; Miura, T.; Morita, T.; Kato, H.;Fujikawa, K.; Iwanaga, S.; Takada, K.; Kimura, T.; Sakakibara, S.,Highly Sensitive Peptide-4-Methylcoumaryl-7-Amide Substrates forBlood-Clotting Proteases and Trypsin. European Journal of Biochemistry1988, 172, (1), 17-25; Stone, S. R.; Hofsteenge, J., Kinetics of theInhibition of Thrombin by Hirudin. Biochemistry 1986, 25, (16),4622-4628.)

FIG. 1( a) is the simulation of the amplification process used inamplification example 1, with set initial concentration of theengineered prothrombin (1.4*10⁻⁶ M), thrombin (1.4*10⁻¹⁰ M), and hirudin(1*10⁻⁸ M), and varied concentration of input which is thrombin. Time ofresponse for each concentration of input was defined as the time whenconcentration of thrombin reaches 80% of the initial concentration ofprothrombin (if this time is larger than 10000 seconds, it was set to10000 seconds). Rate of cleavage of engineered prothrombin by thrombinwas taken to be the same as the rate of cleavage of factor V bythrombin.

FIG. 1( b) is the simulation of the amplification process used inexample 2, with set initial concentration of the factor XII (1×10⁻⁶ M),factor XIIa (1×10⁻¹⁰ M), ecotin (1×10⁻⁷ M), and Boc-Gln-Gly-Arg-MCA(fluorogenic substrate for factor XIIa), and varied concentration ofinput which is kallikrein. Time of response for each particularconcentration of input was defined as the time when concentration of thefluorescent molecules reaches 80% of the initial concentration of thefluorogenic substrate. If this time is larger than 10000 seconds, it wasset to 10000 seconds.

FIG. 1( c) Simulation of the amplification process used in example 4,with set initial concentration of the factor XII (1×10⁻⁶ M), factor XIIa(1×10⁻¹⁰ M), prekallikrein (1×10⁻¹⁰ M), kallikrein (1×10⁻¹⁴ M), ecotin(1×10⁻⁷ M), and Boc-Gln-Gly-Arg-MCA (fluorogenic substrate for factorXIIa), and varied concentration of input which is kallikrein. Time ofresponse for each particular concentration of input was defined as thetime when concentration of the fluorescent molecules reaches 80% of theinitial concentration of the fluorogenic substrate. If this time islarger than 10000 seconds, it was set to 10000 seconds.

FIG. 15 is the experimental results showing how blood clotting timevaried with size of patch of tissue factor, an input for the bloodclotting network discussed in example 6. (Kastrup, C. J.; Runyon, M. K.;Shen, F.; Ismagilov, R. F., Modular chemical mechanism predictsspatiotemporal dynamics of initiation in the complex network ofhemostasis. Proceedings of the National Academy of Sciences of theUnited States of America 2006, 103, (43), 15747-15752.) The patch sizein these experiments correlated with local concentration of tissuefactor.

Amplification Example 7

Amplification process: This reaction network with positive feedbackinvolves proteins in apoptosis. This network includes the production ofcaspase-9 from procaspase-9 catalyzed by cytochrome C and Apaf1, thedimerization of caspase-9, the production of caspase-3 from procaspase-3catalyzed by caspase-9-dimer, the production of caspase-9 fromprocaspase-9 by catalyzed by either caspase-9 dimer or caspase-3.

Inhibitory mechanism: Inhibitors or antibodies for caspase-3 and/orcaspase-9 are used.

Activation mechanism: Similar to examples 1-5, the analyte, bacterialphosphatase, may cleave tagged molecules and release them. Thesemolecules may be caspase-3 and/or caspase-9, or inhibitors of theinhibitors used in the inhibitory mechanism. The specificity of thismethod may be designed to match expectation by changing the specificityof the cleavage of the tag by bacterial phosphatase. If detection of anenzyme different from phosphatase is needed, a different tag is used.

Readout process: caspase-3 and/or caspase-9 enzymatically cleavefluorogenic substrates to give fluorescent signal.

Using the techniques described in this patent, this method is predictedto be feasible with concentrations of enzyme precursors from 0.1 μM to10 μM, be activated with concentration of input after concentratingtechniques of as low as 1 nM, give response after 1-10 minutes, and giveamplification gain of 3 to 5 orders of magnitude.

Amplification Example 8

Limulus amebocyte lysate (LAL) is known to coagulate when bacteriallipopolysaccharide (LPS) is present. One mechanism is the binding of LPSto an 82-kDa protein (termed LPS-binding protein (LBP)), which normallynegatively regulates coagulation. (Roth, R. I.; Tobias, P. S.,Lipopolysaccharide-Binding Proteins of Limulus Amebocyte Lysate.Infection and Immunity 1993, 61, (3), 1033-1039.)

Amplification process: The clotting network of LAL.

Inhibitory mechanism: A small excess of LBP.

Activating mechanism: Generally, the input may be or promote theproduction of some substance that binds to LBP. The input may bebacterial LPS or bacterial phosphatase that may cleave tagged (andinactive) LPS.

Readout process: Clotting or absorbance at 405 nm may be used.

The variations below can be applied individually or in combinations withother variations to all of amplification examples 1-8 shown above.

Variation 1:

Negative contrast is used instead of positive contrast.

Amplification processes: The processes used in examples 1-8 are usedhere.

Inhibitory mechanism: There is no inhibitory mechanism.

Activating mechanism: Inhibitory mechanism used in examples 1-8 are usedas input to see the contrast.

Readout process: The processes used in examples 1-8 are used here.

Variation 2:

An enzyme precursor (inactive form) and an enzyme (active form) do nothave to be two totally different molecules. They only need to havedifferent reactivity.

The inactive form may have an inactive conformation while the activeform has an active conformation. A conformation change may befacilitated by binding of a small molecule to an enzyme, an enzyme toand enzyme, a small molecule to a DNA or RNA molecule, an enzyme to aDNA or RNA molecule, or binding of more than two substances.

The inactive form may be tagged with an inhibitor, thus beingself-inhibitory. When the linker to the inhibitor is cleaved, themolecule is now active. Positive feedback can be incorporated in thesevariations because the active enzyme can catalyze the change ofconformation or the cleavage of the self-inhibiting tag of anotherenzyme of the same kind or of different kind.

Variation 3:

Detection of molecules other than enzymes may be achieved as well. Tothe systems in examples 1-8 without or with any single or combination ofvariations, one or multiple steps is added. The analyte, which may notbe an enzyme, promotes the activation of an enzyme that may cleave thetag from the substrate used in activating mechanisms in examples 1-8,through one or multiple reactions. For example, activation of enzymesmay be done through change of conformation (a result of binding of somemolecule to an enzyme), linking two inactive components to make anactive substance, or activating another enzyme that may activate theenzyme of interest.

Variation 4:

The techniques described above with or without any variations may beused in combinations with amplification using materials and activationmechanism involving generation of high local concentration.

Amplification Example 9

Amplification process: The reaction of the purple complex of Co(III) and2-(5-bromo-2-pyridylazo)-5[N-n-propyl-N-(3-sulfopropyl)amino]phenol(5-Br-PAPS) and oxone has Co²⁺ (aq) as the autocatalyst. (Endo, M.; Abe,S.; Deguchi, Y.; Yotsuyanagi, T., Kinetic determination of tracecobalt(II) by visual autocatalytic indication. Talanta 1998, 47, (2),349-353; Endo, M.; Ishihara, M.; Yotsuyanagi, T., Autocatalyticdecomposition of cobalt complexes as an indicator system for thedetermination of trace amounts of cobalt and effectors. Analyst 1996,121, (4), 391-394; Tsukada, S.; Miki, H.; Lin, J. M.; Suzuki, T.;Yamada, M., Chemiluminescence from fluorescent organic compounds inducedby cobalt(II) catalyzed decomposition of peroxomonosulfate. AnalyticaChimica Acta 1998, 371, (2-3), 163-170.)

Inhibitory mechanism: A ligand that can be used to capture Co²⁺. As atechnical detail, the Co(III).(5-Br-PAPS) complex may be separated fromoxone by immobilizing them into two different layers or keeping themixture dry.

Activating mechanism: The input, which may be an enzyme or a smallmolecule, may cleave or compete for the ligand that captures Co²⁺discussed in the inhibitory mechanism. Alternatively, the input may alsobe a reducing agent that reduces Co³⁺ to Co²⁺ rapidly but reduces oxonemore slowly.

Readout process: The Co(III).(5-Br-PAPS) complex has a purple color thatis lost when the reaction gets activated because of the conversion ofCo(III) into Co(II) and the oxidation of the organic ligand.

Amplification Example 10

Amplification process: The reduction of aqueous Ag(I) is catalyzed byits product, Ag(0). Other metals (such as Pd, Au, and Pt) may also beused for the autocatalytic reduction of metal cations to lower oxidationstates.

Inhibitory mechanism: The reductant may be chemically protected. Forexample, the reductant may be based on hydroquinone, with the hydroxylgroups protected by a group that is removed by the process of interest.The metal cation may be captured by a ligand. The autocatalyst metal maybe coated with small molecules, gel or polymer.

Activating mechanism: The input (enzyme or small molecule) may deprotectthe reductant by cleaving off the protective rings. The input may alsodestroy or compete for the ligand. The input may also remove the coatinglayer on the metal particle.

Readout process: Visual readout may be achieved via aggregates(precipitates) of metal, specific indicator for different oxidationstates of the metals, or general reduction-oxidation indicator.

Amplification Example 11

Amplification process: The reduction-oxidation reaction between chloriteand iodide has iodine as the autocatalyst. (Dateo, C. E.; Orban, M.;Dekepper, P.; Epstein, I. R., Systematic Design of Chemical Oscillators0.5. Bistability and Oscillations in the Autocatalytic Chlorite IodideReaction in a Stirred-Flow Reactor. Journal of the American ChemicalSociety 1982, 104, (2), 504-509.)

Inhibitory mechanism: A compound may be used to consume theautocatalyst, iodine, thus slowing down the reaction effectively to thedegree that no significant output is visualized after a long time.

Activating mechanism: The input may be iodine, or produce iodine throughone or multiple reactions. The input may also be a compound thatinhibits the consumption of iodine, or may produce such compound afterone or many steps.

Readout process: Starch may be used to give a strongly colored bluecomplex with the product iodine.

Amplification Example 12

Amplification process: The reduction-oxidation reaction betweenthiosulfate and chlorite is autocatalytic in both hydronium ion andchloride ion, (Runyon, M. K.; Johnson-Kerner, B. L.; Ismagilov, R. F.,Minimal functional model of hemostasis in a biomimetic microfluidicsystem. Angewandte Chemie-International Edition 2004, 43, (12),1531-1536.

Horvath, A. K.; Nagypal, I.; Epstein, I. R., Kinetics and mechanism ofthe chlorine dioxide-tetrathionate reaction. Journal of PhysicalChemistry A 2003, 107, (47), 10063-10068; Horvath, A. K.; Nagypal, I.;Epstein, I. R., Oscillatory photochemical decomposition of tetrathionateion. Journal of the American Chemical Society 2002, 124, (37),10956-10957; Nagypal, I.; Epstein, I. R., Systematic Design of ChemicalOscillators 0.37. Fluctuations and Stirring Rate Effects in the ChloriteThiosulfate Reaction. Journal of Physical Chemistry 1986, 90, (23),6285-6292.) and may be activated by Ag⁺ which is predicted to oxidizethiosulfate and release hydronium or chloride (preliminary result).

Inhibitory mechanism: The amount of hydronium ion may be kept in checkby using a pH buffer. Chloride anion may be sequestered by a cation thatis a sufficiently weak oxidizer to not react with thiosulfate. Ag⁺ maybe sequestered by ligands. Solids that produce an acidic environment orsolids that are salts of Cl⁻ or Ag⁺ may be coated with materials such asgel or polymer.

Activating mechanism: The input may be acidic or promote the productionof any acid through one or multiple steps. The input may also bechloride anion or promote the production of chloride anion, or competewith the cation that captures chloride anion, or react with the cationto disable its ability to capture chloride anion. The input may also beAg⁺ or produce Ag⁺ by compete for or inactivate the ligands for Ag⁺. Theinput may also break the coating materials of the solid particles ifsuch inhibitory mechanism is used.

Readout process: A pH or reduction-oxidation indicator may be used.

Using the techniques described in this patent and the reactionconditions previously described, (Kastrup, C. J.; Runyon, M. K.; Shen,F.; Ismagilov, R. F., Modular chemical mechanism predicts spatiotemporaldynamics of initiation in the complex network of hemostasis. Proceedingsof the National Academy of Sciences of the United States of America2006, 103, (43), 15747-15752.) one can get response in 1-2 minutes.

Amplification Example 13

Amplification process: The organic reactions developed by Ichimura andcoworkers (Ichimura, K., Nonlinear organic reactions to proliferateacidic and basic molecules and their applications. Chemical Record 2002,2, (1), 46-55.) have acids as autocatalysts.

Inhibitory mechanism: The amount of hydronium ion may be kept in checkby using a pH buffer or a weak base.

Activating mechanism: The input may be acidic or promote the productionof any acid through one or multiple steps.

Readout process: A pH or reduction-oxidation indicator is used.

The variations below can be applied individually or in combinations withother variations to all of amplification examples 9-13 shown above

Variation 1:

Negative contrast is used instead of positive contrast.

Amplification processes: The processes used in examples 1-5 describedabove are used.

Inhibitory mechanism: There is no inhibitory mechanism.

Activating mechanism: Inhibitory mechanism used in examples 1-5described above are used as input to see the contrast.

Readout process: The processes used in examples 1-5 described above areused here.

Variation 2:

The techniques described above with or without any variation may be usedin combinations amplification using materials and activation mechanisminvolving generation of high local concentration.

Amplification Example 14

Provided the analytes are particles that can be tagged with someactivating molecules through various methods (such as antibodies,His-tag/Ni/NTA, biotin/avidin etc), a method can be used to detect smallconcentration of the analytes in which the amplification process may beany. The activating molecule is first concentrated on particles. Thenstochastic confinement is performed to concentrate these particles intoplugs. The concentrated activating molecules act as input for theamplification process. Because excess activating molecules are notconcentrated on particles, even after stochastic confinement, theconcentration is still not high enough to activate the amplificationprocess.

For example, the amplification process may be chosen as the onedescribed in example 5 of section 1 and in FIG. 13, the activatingmolecules may be chosen to be InhA1, as shown in FIG. 16. The particularexample is predicted to be able to detect particles of concentration ofas low as 1fM, which may hypothetically be amplified to 1 nM afterstochastic confinement. To total time of detection is predicted to be1-10 minutes.

FIG. 16 illustrates combining amplification cascades with stochasticconfinement enables sensitive detection of single particles or more. Thegeneral idea is described in the text. The protease shown hererepresents an activating molecule in general. (A and B) Particles areadded to a container containing the activating molecules. (C) Theparticles bind to the activating molecules and are stochasticallyconfined in plugs. (D) In plugs loaded with particles, the concentrationof activating molecules is above the detection threshold and produces athreshold signal, while plugs without particles do not. (E and F) Insolutions with excess but sub-threshold concentrations of activatingmolecules, stochastic confinement of particles into plugs (G) results ina few plugs containing a particle with multiple copies of the activatingmolecules attached and many plugs with a few copies of activatingmolecules. (H) Plugs containing a particle initiate the cascade, becausestochastic confinement of the particles has concentrated the activatingmolecules in occupied plugs, whereas plugs without a particle remain atthe sub-threshold protease concentration of the bulk solution.

Amplification Example 15

The analytes are particles which bind to activating molecules. Thereactions may be chosen from those described in sections 1 and 2. Forexample, if the system with example 2 in section 1 is chosen, theactivating molecule is kallikrein.

Amplification process: The enzyme precursor is factor XII that may becleaved by factor XIIa to produce more factor XIIa in the presence ofdextran sulfate or negatively charge surface in general.

Suppose a sample containing low concentration of type-B particles (suchas 100 CFU/mL) has a large excess of interfering type-A particles (suchas 10⁵ CFU/mL) that can bind much less activating molecules per particle(such as 100 fold) (with a specifically designed antibody or any othermeans), but much more collectively. Under bulk detection mechanisms,either with amplification or by classical methods for measuring enzymeconcentrations, the activity due to the excess of type-A particles willdominate the response (FIGS. 17 A and B). When the tagged particles arestochastically confined in plugs, only those containing type-B particleswill have enough activating molecules to activate the amplificationprocess. In general, any kind of particles which can bind to any ofactivating molecules described in sections 1 and 2 through antibodies orany other method may be detected using this method. For example, thistechnique may be use to detect B. anthracis from a sample containing alot of interfering bacteria such as B. circulans and other kinds.

FIG. 17 illustrates selective detection of particles by using stochasticconfinement. (A and B) In a bulk solution containing both a highconcentration of interfering type-A particles (such as the bacteria B.circulans) (small gray particles binding few activating molecules (suchas kallikrein)) and a low concentration of target type-B particles (B.anthracis) (large gray particles binding many activating molecules), theactivity of the excess type-A particles dominates. Therefore, solutionswith excess type-A particles (A) and excess type-B particle with a lowconcentration of type-A particles (B) both trigger a detection response(dark gray) (C and D). When stochastically confined in plugs, the amountof activating molecule in each plug made from solution A remains belowthe detectible level, while the amount of activating molecules of plugscontaining type-B particles from solution B results in readout of type-Bparticles (dark gray plug), not from interfering type-A particles(lighter gray plugs).

Amplification Example 16

This technique is a variation of example 2 in section 2. Ag particlesare coated with tags to form Ag—(X—Y)_(n). This coated particle cannotbind with bacteria. However, the input cleaves or produces somesubstance that cleaves X—Y, exposing X on the surface of the particles.These particles now can be locally concentrated using techniques shownin section 4ii.

Amplification Example 17

This technique is a variation of example 4 in section 2. This systemcontains AgCl particles coated with an inert shell (such as Ca₃(PO₄)₂)and protected ethylenediaminetetraacetic acid (EDTA) ligands (in thesolution or in the particle), and thiosulfate and chlorite in thesolution. The input deprotects EDTA, allowing it to complex with Ca²⁺,dissolving the inert shell, exposing AgCl to the thiosulfate/chloritemixture and activates the amplification process. The threshold is set upby the thickness of the inert shell. To detect different kinds of input,the method to protect and deprotect EDTA may be customized. The majorreaction in this amplification process was described previously.(Runyon, M. K.; Johnson-Kerner, B. L.; Ismagilov, R. F., Minimalfunctional model of hemostasis in a biomimetic microfluidic system.Angewandte Chemie-International Edition 2004, 43, (12), 1531-1536;Horvath, A. K.; Nagypal, I.; Epstein, I. R., Kinetics and mechanism ofthe chlorine dioxide-tetrathionate reaction. Journal of PhysicalChemistry A 2003, 107, (47), 10063-10068; Horvath, A. K.; Nagypal, I.;Epstein, I. R., Oscillatory photochemical decomposition of tetrathionateion. Journal of the American Chemical Society 2002, 124, (37),10956-10957; Nagypal, I.; Epstein, I. R., Systematic Design of ChemicalOscillators 0.37. Fluctuations and Stirring Rate Effects in the ChloriteThiosulfate Reaction. Journal of Physical Chemistry 1986, 90, (23),6285-6292.)

1. A method of detecting bacteria in a sample, comprising: creating anarray of plugs by introducing a first plug fluid into a flow of carrierfluid in a microchannel; wherein the majority of plugs in the array donot contain a bacterium; wherein the first plug fluid is substantiallyimmiscible with the carrier fluid and comprises a concentration of thesample diluted such that at most 2 bacteria are present in any plug; andanalyzing the array for the presence of bacteria.
 2. The method of claim1, wherein the array is analyzed for a detectable signal produced by thebacteria, wherein the detectable signal is a substance produced by thebacteria or is produced when the bacteria consumes a substance in theplug.
 3. The method of claim 2, wherein the substance is selected fromthe group consisting of oxygen, carbon, a protein produced by thebacteria, a molecule produced by a bacterial enzymatic reaction, and aredox/potential sensitive indicator. 4-5. (canceled)
 6. The method ofclaim 1, wherein the sample is from human, soil or marine.
 7. (canceled)8. The method of claim 1, wherein the bacteria are at a higherconcentration in the plugs than in the sample.
 9. The method of claim 1,wherein the plugs contain different species of bacteria.
 10. The methodof claim 1, further comprising introducing a plug fluid comprising mediacapable of supporting bacterial growth into the plug.
 11. The method ofclaim 10, wherein the detectable signal is produced by growth of thebacteria.
 12. The method of claim 1, wherein the plugs comprise asubstance capable of inducing virulence in the bacteria.
 13. The methodof claim 1, wherein the at least two plug comprise a substance capableof lysing the bacteria.
 14. (canceled)
 15. The method of claim 1,further comprising assaying the bacteria detected for a biologicalactivity.
 16. The method of claim 1, further comprising identifying thebacteria.
 17. The method of claim 1, further comprising splitting a plugthat has been determined to contain bacteria into multiple plugs, eachcontaining at least one of the bacteria.
 18. The method of claim 1,further comprising conducting a polymerase chain reaction on thecontents of the at least two plugs prior to analyzing the at least twoplugs for the detectable signal.
 19. A method of detecting bacteriacomprising: flowing at least two plugs in a carrier fluid through amicrochannel; wherein each plug comprises a plug fluid substantiallyimmiscible with the carrier fluid; wherein a first plug comprises ameans for detecting a first species of bacteria; wherein a second plugcomprises a means for detecting a second species of bacteria differentfrom the first species of bacteria; introducing a sample optionallycomprising bacteria into the first and second plugs, wherein thebacteria produce a detectable signal; and analyzing the plugs for thedetectable signal. 20-28. (canceled)
 29. A method of screening forantibiotic activity comprising: flowing at least two plugs in a carrierfluid through a microchannel; wherein each plug comprises a plug fluidimmiscible with the carrier fluid and media capable of supportingbacterial growth; wherein the first plug comprises a first antibioticcandidate; wherein the second plug comprises a second antibioticcandidate; introducing a sample which comprises bacteria into the firstand second plugs; and detecting the presence of bacterial growth. 30-44.(canceled)
 45. A method of detecting bacteria in an aqueous sample,comprising: dividing a sample into a plurality of subsamples in amicrofluidic device, where each subsample is separated from another by afluorinated liquid, each subsample has a volume that is a nanoliter orless, the majority of the subsamples do not contain a bacterium, andeach subsample has at most 2 bacteria; and analyzing the volumes for thepresence of bacteria.